Methods and materials relating to CD39-like polypeptides

ABSTRACT

The invention provides novel polynucleotides isolated from cDNA libraries of human fetal liver-spleen and macrophage as well as polypeptides encoded by these polynucleotides and mutants or variants thereof. The polypeptides correspond to a novel human CD39-like protein. Other aspects of the invention include vectors containing polynucleotides of the invention and related host cells as well a processes for producing novel CD39-like polypeptides, and antibodies specific for such polypeptides.

1. RELATED APPLICATIONS

[0001] This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 09/481,238 filed Jan. 11, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/370,265filed Aug. 9, 1999 which is a continuation-in-part of PCT patentapplication Serial No. PCT/US99/16180 filed Jul. 16, 1999 which is acontinuation-in-part of U.S. patent application Ser. No. 09/350,836filed Jul. 9, 1999 which is a continuation-in-part of U.S. patentapplication Ser. No. 09/273,447 filed Mar. 19, 1999 which is acontinuation-in-part of U.S. patent application Ser. No. 09/122,449filed Jul. 24, 1998 and also a continuation-in-part of U.S. patentapplication Ser. No. 09/244,444 filed Feb. 4, 1999, which in turn is acontinuation of U.S. patent application Ser. No. 09/118,205 filed Jul.16, 1998, the disclosures of all of which are incorporated by referenceherein in their entirety.

2. FIELD OF THE INVENTION

[0002] This invention relates in general to novel polynucleotidesisolated from cDNA libraries of human fetal liver-spleen and macrophagesand to polypeptides encoded by these polynucleotides. In particular, theinvention relates to a human CD39-like protein with homologies to ATPdiphosphohydrolases and variants thereof.

3. BACKGROUND

[0003] CD39 (cluster of differentiation 39) is a cell-surface moleculerecognized by a “cluster” of monoclonal antibodies that can be used toidentify the lineage or stage of differentiation of lymphocytes and thusto distinguish one class of lymphocytes from another. This CD39 moleculewas originally defined as a B lymphocyte marker (Rowe, M., et al. Int.J. Cancer 29:373 (1982)). Subsequent studies have shown CD39 to be amarker for a distinct subset of activated lymphocytes within theallosensitized CD8-positive cytotoxic cells (Gouttefangeas C., et al.,Eur. J. Immunol. 22:2681 (1992)). Outside of lymphoid tissue, CD39 canbe found in quiescent vascular endothelial cells (Kansas, G. S., et al.,J. Immunol. 146:2235 (1991)) and throughout rat brain in the neurons ofthe cerebral cortex, hippocampus, and cerebellum, as well as in glialcells (Wang, T-F. and Guidotti, G., Brain Res. 790:318 (1998)).

[0004] CD39 is a 510-amino acid protein with a predicted molecular massof 57 kDa. However, because of heavy glycosylation at asparagineresidues (six potential N-glycosylation sites) the molecule displays amobility closer to 100 kDa (Maliszewski, C. R., et al., J. Immunol.153:3574 (1994)). CD39 contains two hydrophobic regions, one near theamino terminus and the other near the carboxyl terminus which arebelieved to be transmembrane regions.

[0005] The role of CD39 in platelet aggregation and ATP/ADP hydrolysisis unclear. Although CD39 was originally reported to be an ectoADPasewith a preference for ATP over ADP as a substrate, Wang, et al., J.Biol. Chem. 271:9898-9901(1996), Marcus, et al., J. Clin. Invest.99:1351-1360 (1997) reported that CD39 was unique for its highpreference for ADP over ATP as a substrate and in 1998, Gayle, et al.,J. Clin Invest. 10:1851-1859 (1998), described CD39 as an ectoADPasewith no preference for one substrate over the other.

[0006] Reports that several ATP Diphosphohydrolases (ATPDases) shareamino acid sequence homology with CD39 have been substantiated by theshowing that CD39 is itself an ATPDase (Wang, T- F., et al., J. Biol.Chem. 271:9898 (1996); Kaczmarek, E., et al., J. Biol. Chem. 271:33116(1996)). Since CD39 is a plasma membrane-bound enzyme, CD39 has beentermed an “ecto-ATPase,” but CD39 is more often referred to as an“ecto-apyrase” because of the reduced rate of hydrolysis of ADP whencompared with ecto-ATPases.

[0007] This activity has shown to modulate platelet reactivity andaggregation in response to vascular injury. During vascular injury,activated platelets aggregate forming an occlusive thrombus. Excessiveplatelet accumulation at sites of vascular injury can contribute tovessel occlusion. Endothelial cells respond to the potentially occlusiveeffects of platelet aggregation by several mechanisms. One of thesemechanisms results ecto-apyrase-mediated removal of ADP, which in turneliminates platelet reactivity and recruitment. It is now known that theendothelial ecto-apyrase responsible for this ADP removal is CD39(Marcus, A. J., et al., J. Clin. Invest. 99:1351 (1997)).

[0008] Recently, CD39 was engineered to produce a soluble form of themolecule. This soluble CD39 was shown to display the same nucleotidaseactivity as the membrane-bound molecule (Gayle, R. B., et al., J. Clin.Invest. 101:1851 (1998)). Intravenously administered soluble CD39 alsoremained active in mice for an extensive period of time, indicating thatsoluble CD39 could be useful as a inhibitor of platelet aggregation inthe prophylaxis or treatment of platelet-mediated thrombotic conditions.

[0009] Platelet aggregation inhibitors (antithrombotic agents) decreasethe formation or the action of chemical signals that promote plateletaggregation. Currently available antithrombotic agents include aspirin,ticlopidine, and dipyridamole. These agents have proven beneficial inthe prevention and treatment of occlusive cardiovascular diseases,including myocardial infarction, cerebral ischemia, angina.Antithrombotic therapy has also been used in the maintenance of vasculargrafts.

[0010] Myocardial infarction is the development of necrosis of themyocardium (the middle muscular layer of the heart wall) due to acritical imbalance between oxygen and myocardial demand. The most commoncause of acute myocardium infarction is narrowing of the epicardialblood vessels due to atheromatous plaques. Plaque rupture withsubsequent exposure of basement membrane results in platelet aggregationand thrombus formation, which can result in partial or completeocclusion of the vessel and subsequent myocardial ischemia.

[0011] In cerebral ischemia, inadequate blood flow results from anocclusion in a blood vessel or hemorrhaging. In the latter case,excessive bleeding in one area of the brain deprives another area ofblood. If the damage occurs in a singular small area, “transient” or“focused” cerebral ischemia results. When a major artery is blocked(carotid artery) global or diffused ischemia results. The primarymedical strategy for secondary prevention of stroke is antiplatelettherapy. Aspirin is currently employed for reducing the risk ofrecurrent transient ischemic attacks or stroke in men who have transientischemia of the brain due to fibrin emboli.

[0012] Each year, thousands of patients suffer a decline in blood flowto one or more limbs. Without sufficient blood flow, and, unless bloodflow can be restored in time, the limb must be amputated. In some cases,grafts from the patient's veins can be used to form new arteries.However, in cases where the quality of the veins is poor, polymericvascular grafts are typically used. The polymeric grafts are inherentlythrombogenic as the blood constituents passing through the grafts becomeactivated and tend to form clots. Efforts to line the grafts withendothelial cells can reduce blood clotting, but better results areobtained when antithrombotic therapy is employed.

[0013] Angina pectoris is a characteristic chest pain caused byinadequate blood flow through the blood vessels of the myocardium. Theimbalance between oxygen delivery and utilization may result from aspasm of the vascular smooth muscle or from obstruction of blood vesselscaused by atherosclerotic lesions. Three classes of drugs have beenshown to be effective in treating angina: nitrates, beta-blockers andcalcium channel blockers. Currently, the antithrombotics dipyridamoleand aspirin are employed to prophylactically treat angina pectoris.

[0014] Ecto-apyrases, such as CD39, offer a number of advantages overseveral of the standard antithrombotics. For example, aspirin treatmentcontrols the prothrombotic action of thromboxane; however, aspirin alsoprevents formation of antithrombotic prostacyclin, which limitsaspirin's efficacy. Another antithrombotic, endothelium-derived relaxingfactor (nitric oxide; “EDRF/NO”), is inhibited in vitro and in vivo byhemoglobin after its rapid diffusion into erythrocytes. In contrast,CD39 is aspirin-insensitive and completely inhibits platelet reactivityeven when eicosanoid and EDRF/NO production are blocked.

[0015] CD39's ATPDase activity also implicates CD39 in the modulation ofneurotransmission. ATP is a major purinergic neurotransmitter that isoften co-released into the synaptic cleft with severalneurotransmitters. Responses to ATP are mediated by specific plasmamembrane receptors, called P2 purinergic receptors (Dubyak, G. R. andEl-Motassim,C. Am J. Physiol. 34:C577-C606 (1993)). The distribution ofCD39 in the rat brain indicates that CD39 plays a role in terminating P2purinergic neurotransmission (Wang, T. F. and Guidotti, G., Brain Res.790:318 (1998)). Furthermore, a decrease in ecto-apyrase activity isbelieved to lead to an accumulation of the excitatory neurotransmitter,extracellular ATP, as well as a deficiency of the endogenousanticonvulsant extracellular adenosine.

[0016] The chomosomal localization of CD39 provides additional supportfor a role in modulation of neurotransmission. More specifically, CD39has been mapped to chromosome 10q 23.1-24.1 (Maliszewski, C. R., et al.,J. Immunol. 153:3574 (1994)), and this site overlaps with thesusceptibility locus for human partial epilepsy with audiogenic symptoms(Ottman, R. et al., Nature Genet. 10:56 (1995)). This co-localization ofthe CD39 gene and the susceptibility locus has led to the hypothesisthat decrease in ecto-apyrase activity in the brain is the primary causeof partial epilepsy (Wang T-F., et al., Mol. Brain Res. 47:295 (1997)).

[0017] A screen for human cDNAs that hybridize to cosmids from the humanchromosome 9q34 region lead to the identification of a transcript withhigh homology to a chicken muscle ecto-ATPase (60% identity) and theecto-apyrase CD39 (41% amino acid identity) (Chadwick, B. P., Mamm.Genome 8:668 (1997)). This gene, designated “CD39-like-1 gene” (CD39L1),has a higher degree of homology to CD39 than does chicken muscleecto-ATPase. The biological activity of this protein has not been testedbut on the basis of the high amino acid homology, CD39L1 is believed tobe a new member of the ecto-ATPase family. Recently, a mouse gene withhomology to NTPases was cloned and sequenced (Acc. No. AF006482) byChadwick et al. (Mamm. Gen. 9:162-164 (1998).)

4. SUMMARY OF THE INVENTION

[0018] The invention is based on polynucleotides isolated from cDNAlibraries prepared from human fetal liver-spleen and macrophages. Thecompositions of the present invention include novel isolatedpolypeptides with apyrase and/or NDPase activity, in particular, novelhuman CD39-like polypeptides, and active variants thereof, isolatedpolynucleotides encoding such polypeptides, including recombinant DNAmolecules, cloned genes or degenerate variants thereof, especiallynaturally occurring variants such as allelic variants, antisensepolynucleotide molecules, and antibodies that specifically recognize oneor more epitopes present on such polypeptides, as well as hybridomasproducing such antibodies.

[0019] The compositions of the invention additionally include vectors,including expression vectors, containing the polynucleotides of theinvention, cells genetically engineered to contain such polynucleotidesand cells genetically engineered to express such polynucleotides.

[0020] The isolated polynucleotides of the invention include naturallyoccurring or wholly or partially synthetic DNA, e.g., cDNA and genomicDNA, and RNA, e.g., mRNA. One polynucleotide according to the inventionencodes a novel CD39-like protein having the amino acid sequence shownin FIG. 2 (SEQ ID NO. 3), which has been designated CD39-L4. Anotherpolynucleotide according to the invention encodes a novel CD39-likeprotein having the amino acid sequence shown in SEQ ID NO: 27, which hasbeen designated CD39-L2. In another embodiment, a polynucleotideaccording to the invention encodes a novel CD39-like protein having thefull length or mature amino acid sequence set forth in SEQ ID NO. 25,which has been designated CD39-L66, and is an isoform of CD39-L4. Theisolated polynucleotides of the invention include a polynucleotidecomprising the nucleotide sequence of SEQ ID NO. 2, 24 or 26. Thepolynucleotides of the invention also include polynucleotides thatencode polypeptides with a biological activity of the polypeptide of SEQID NO. 3 or 27 (including apyrase or NDPase activity) such as (a) thenucleotide sequence of SEQ ID NO. 2, 24, 26 or (b) a nucleotide sequenceencoding the full length or mature amino acid sequence of SEQ ID NO. 3,25, or 27; (c) a polynucleotide which is an allelic variant of anypolynucleotide recited above; (d) a polynucleotide that hybridizes understringent conditions to (a) or (b); (e) or a polynucleotide that encodesa polypeptide comprising at least one CD39-like domain, e.g. catalyticdomain.

[0021] The polynucleotides of the invention additionally include thecomplement of any of the polynucleotides recited above.

[0022] A collection as used in this application can be a collection ofonly one polynucleotide. The collection of sequence information oridentifying information of each sequence can be provided on a nucleicacid array. In one embodiment, segments of sequence information areprovided on a nucleic acid array to detect the polynucleotide thatcontains the segment. The array can be designed to detect nucleic acidsthat are perfectly complementary (full-match) or mismatched to thepolynucleotide that contains the segment. The collection can also beprovided in a computer-readable format.

[0023] The invention also provides a polynucleotide including anucleotide sequence that is substantially equivalent to thesepolynucleotides. Polynucleotides according to the invention can have atleast about 80%, more typically at least about 90%, and even moretypically at least about 95%, sequence identity to a polynucleotide ofSEQ ID NO. 2, 24 or 26 and specifically include a human polynucleotidewhich has at least 80% sequence identity to a polynucleotide of SEQ IDNO. 2, 24 or 26; or a polynucleotide which has at least 90% sequenceidentity to a polynucleotide of SEQ ID NO. 2, 24 or 26. Similarly,polypeptides of the invention include polypeptides having apyrase orNDPase activity and at least about 80%, 90% or 95% sequence identity toSEQ ID NO. 3, 25 or 27. Polypeptides of the invention further includemultimeric, especially dimeric, polypeptides having apyrase or NDPaseactivity and at least about 80%, 90% or 95% sequence identity to SEQ IDNO. 3, 25 or 27.

[0024] A further aspect of the invention is the development of novelCD39-L4 polynucleotide or polypeptide variants which preferably exhibitincreased recombinant expression levels or improved ADPase or NDPaseactivity compared to wild type CD39-L4 (SEQ ID NO: 5). This aspect ofthe invention includes polypeptides comprising at least one amino acidsubstitution selected from the group consisting of: D168-T, S170-Q andL175-F, wherein said substitution(s) result in increased ADPase activityof the polypeptide. One preferred embodiment is the polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 7 (encoded by thenucleotide sequence of SEQ ID NO. 6), which is a variant CD39-L4containing all three substitutions that has been designated ACRIII. Aplasmid containing this DNA was deposited with the American Type CultureCollection (ATCC), 10801 University Avenue, Manassas, Va., on Jul. 13,1999 under the terms of the Budapest Treaty (ATCC accession numberPTA-346). Alternatively, instead of making the specific D168-T, S170-Qand/or L175° F. substitution(s), substitution of amino acids withsimilar properties is contemplated. Additional conservativesubstitutions at amino acid positions other than D168, S170 and/or L175are further contemplated. For example, all of the corresponding aminoacids from CD39 could be substituted for amino acids 167-181 of CD39-L66or CD39-L4.

[0025] This aspect of the invention also specifically contemplates that,in view of the fact that variant polynucleotides containing changes inthe codons for amino acid 168, 170 and 175 are more highly expressed,such polynucleotides may be more highly expressed if the codons at thesesame positions are modified without changing the wild type amino acidsequence (e.g., polynucleotides having codon substitutions at or aroundnucleotide positions 747-749, 753-755, and/or 768-770 of SEQ ID NO: 2,or positions 502-504, 508-510 and/or 523-525 of SEQ ID NO: 4, orcorresponding nucleotide positions in SEQ ID NO: 24 are contemplated).

[0026] In addition, development of novel CD39-L2 polynucleotide orpolypeptide variants which preferably exhibit increased recombinantexpression levels or improved ADPase or NDPase activity compared to wildtype CD39-L2 (SEQ ID NO: 27) is also contemplated. This aspect of theinvention includes polypeptides comprising at least one amino acidsubstitution wherein said substitution(s) result in increasedrecombinant expression levels or increased ADPase activity of thepolypeptide, as well as polynucleotides encoding the wild type CD39-L2sequence that have silent codon substitutions at nucleotide positions806-808, 812-814 and/or 827-829 corresponding to those identified abovefor CD39-L4.

[0027] Polynucleotides encoding these polypeptides, vectors and hostcells comprising such polynucleotides, methods of using such host cellsto produce polypeptides, and other therapeutic products comprising thepolypeptides (including fusion proteins in which the CD39-likepolypeptide is fused to a heterologous peptide or polypeptide, such asan immunoglobulin constant region, or derivatives in which the CD39-likepolypeptide is modified by water soluble polymers to increase itshalf-life) are also comprehended by the invention, as are methods oftreating a subject suffering from a disorder relating to thrombosis,coagulation or platelet aggregation by administering such therapeuticproducts.

[0028] The invention further comprises methods of inhibiting plateletaggregation in a mammalian subject by reducing the ratio of ADP:ATP in amammalian subject to a less than normal ratio by administering thepolypeptides of the invention or by administering polypeptides withADPase activty and at least about 90% sequence identity to SEQ ID NO: 3,25 or 27. Preferably the ratio of ADP:ATP is reduced withoutsignificantly affecting ATP levels. In one embodiment, the ADP:ATP ratiois reduced systemically in circulation. In another embodiment, theADP:ATP ratio is reduced locally, for example, in heart, brain, kidney,lungs, limbs or other organs.

[0029] Methods of identifying compounds capable of reducing the ratio ofADP:ATP to a less than normal ratio are also contemplated. For example,compounds may be identified by steps including: determining apyraseactivity of said compound on ATP; determining apyrase activity of saidcompounds on ADP; and selecting a compound that has greater activitywith respect to ADP compared to ATP. Exemplary compounds to be screenedinclude, but are not limited to, CD39-L4 and CD39-L2 variants.

[0030] Gene therapy techniques are also provided to modulate diseasestates associated with CD39-L4 or CD39-L2 expression and/or biologicalactivity. Delivery of a functional CD39-L4 or CD39-L2 gene toappropriate cells is effected ex vivo, in situ, or in vivo by use ofvectors, and more particularly viral vectors (e.g., adenovirus,adeno-associated virus, or a retrovirus), or ex vivo by use of physicalDNA transfer methods (e.g., liposomes or chemical treatments).

[0031] The invention also relates to methods for producing polypeptidesof the invention comprising growing a culture of cells of the inventionin a suitable culture medium under conditions permitting expression ofthe desired polypeptide, and purifying the protein from the cells or theculture medium. Preferred embodiments include those in which the proteinproduced by such process is a mature form of the protein.

[0032] Protein compositions of the present invention, includingtherapeutic compositions, comprise polypeptides of the invention andoptionally an acceptable Carrier, such as a hydrophilic (e.g.,pharmaceutically acceptable) carrier.

[0033] Polynucleotides according to the invention have numerousapplications in a variety of techniques known to those skilled in theart of molecular biology. These techniques include use as hybridizationprobes, use as oligomers for PCR, use for chromosome and gene mapping,use in the recombinant production of protein, and use in generation ofanti-sense DNA or RNA, their chemical analogs and the like. For example,because the expression of CD39-L4 and CD39-L2 mRNA is largely restrictedto specific tissues (CD39-L4 in macrophages and CD39-L2 in adult heartand fetal brain), polynucleotides of the invention can be used ashybridization probes to detect the presence of specific mRNA in a sampleusing, e.g., in situ hybridization.

[0034] In other exemplary embodiments, the polynucleotides are used indiagnostics as expressed sequence tags for identifying expressed genesor, as well known in the art and exemplified by Vollrath, et al.,Science 258:52-59 (1992), as expressed sequence tags for physicalmapping of the human genome.

[0035] A polynucleotide according to the invention can be joined to anyof a variety of other nucleotide sequences by well-establishedrecombinant DNA techniques (see Sambrook, J., et al. (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Usefulnucleotide sequences for joining to polypeptides include an assortmentof vectors, e.g., plasmids, cosmids, lambda phage derivatives,phagemids, and the like, that are well known in the art. Accordingly,the invention also provides a vector including a polynucleotide of theinvention and a host cell containing the polynucleotide. In general, thevector contains an origin of replication functional in at least oneorganism, convenient restriction endonuclease sites, and a selectablemarker for the host cell. Vectors according to the invention includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors. A host cell according to the invention can be aprokaryotic or eukaryotic cell and can be a unicellular organism or partof a multicellular organism.

[0036] The polypeptides according to the invention can be used in avariety of conventional procedures and methods that are currentlyapplied to other proteins. For example, a polypeptide of the inventioncan be used to generate an antibody which specifically binds thepolypeptide. The polypeptides of the invention having ATPDase activityare also useful for inhibiting platelet aggregation and can therefore beemployed in the prophylaxis or treatment of pathological conditionscaused by the inflammatory response. The polypeptides of the inventioncan also be used as molecular weight markers, and as a food supplement.

[0037] Another aspect of the invention is an antibody that specificallybinds the polypeptide of the invention. Such antibodies can be eithermonoclonal or polyclonal antibodies, as well fragments thereof andhumanized forms or fully human forms, such as those produced intransgenic animals. The invention further provides a hybridoma thatproduces an antibody according to the invention and anti-idiotypeantibodies.

[0038] Antibodies of the invention are useful for detection and/orpurification of the polypeptides of the invention.

[0039] Methods are also provided for preventing, treating orameliorating a medical condition, including thrombotic diseases, whichcomprises administering to a mammalian subject, including but notlimited to humans, a therapeutically effective amount of a compositioncomprising a polypeptide of the invention or a therapeutically effectiveamount of a composition comprising a binding partner of (e.g., antibodyspecifically reactive for) CD39-like polypeptides of the invention. Themechanics of the particular condition or pathology will dictate whetherthe polypeptides of the invention or binding partners (or inhibitors) ofthese would be beneficial to the individual in need of treatment.

[0040] The invention also provides a method of inhibiting plateletfunction comprising administering a CD39-L4 or CD39-L2 polypeptide ofthe invention to a medium comprising platelets. According to thismethod, polypeptides of the invention can be administered to produce anin vitro or in vivo inhibition of platelet function. A polypeptide ofthe invention can be administered in vivo as antithrombotic agent aloneor as an adjunct to other therapies.

[0041] Also provided are methods of hydrolyzing nucleotide diphosphatescomprising administering CD39-L4 or CD39-L2 polypeptides of theinvention to a medium comprising nucleotidediphosphates. According tothis method, polypeptides of the invention can be administered toproduce an in vitro or in vivo hydrolysis of nucleotidediphosphates. Apolypeptide of the invention can be administered in vivo alone or as anadjunct to other therapies. For example, CD39-L4 or CD39-L2 polypeptidesof the invention may be administered to prevent or treat cancerconditions involving elevated levels of one or more nucleotidediphosphates.

[0042] The invention further provides methods for manufacturingmedicaments useful in the above described methods relating to plateletaggregation and thrombosis.

[0043] The invention also provides methods for detecting or quantitatingthe presence of the polynucleotides or polypeptides of the invention ina tissue or fluid sample, and corresponding kits that comprise suitablepolynucleotide probes or antibodies, together with an optionalquantitative standard. Such methods and kits can be utilized as part ofprognostic and diagnostic evaluation of patients and for theidentification of subjects exhibiting a predisposition to plateletmediated conditions.

[0044] The invention also provides methods for the identification ofcompounds that modulate (i.e. increase or decrease) the expression oractivity of the polynucleotides and/or polypeptides of the invention.Such methods can be utilized, for example, for the identification ofcompounds and other substances that interact with (e.g., bind to) thepolypeptides of the invention, and assays for identifying compounds andother substances that enhance or inhibit the activity of thepolypeptides of the invention, such assays comprising the step ofmeasuring activity of such polypeptides in the presence and absence ofthe test compound.

5. BRIEF DESCRIPTION OF THE DRAWINGS

[0045]FIG. 1 shows polynucleotide sequences according to the invention.SEQ ID NO:1 was obtained from the b2HFLS20W cDNA library using standardPCR, sequencing by hybridization signature analysis, and single pass gelsequencing technology. A-adenosine; C-cytosine; G-guanosine; T-thymine.Ambiguous positions are designated as follows: R indicates A or G; Mindicates A or C; W indicates A or T; Y indicates C or T; S indicates Cor G; K indicates G or T; V indicates A or C or G; H indicates A or C orT; D indicates A or G or T; B indicates C or G or T; and N indicates anyof the four bases.

[0046] SEQ ID NO:2 is an extended version of SEQ ID NO:1 which wasobtained as described in Example 2.

[0047]FIG. 2 shows an amino acid sequence corresponding to thepolynucleotide sequence of SEQ ID NO:2. This sequence is designated asSEQ ID NO:3. The open reading frame encoding SEQ ID NO:3 begins atnucleotide 246 (numbered from the 5′ end) of SEQ ID NO:2. A—Alanine;R—Arginine; N—Asparagine; D—Aspartic Acid; C—Cysteine; E—Glutamic Acid;Q—Glutamine; G—Glycine; H—Histidine; I—Isoleucine; L—Leucine; K—Lysine;M—Methionine; F—Phenylalanine; P—Proline; S—Serine; T—Threonine;W—Tryptophan; Y—Tyrosine; V—Valine; X—any of the twenty amino acids.

[0048]FIG. 3 shows the amino acid sequence alignment of SEQ ID NO:3(identified as “246 prot”) and human CD39 (“CD39Human.seq”). The aminoacid residues are designated as for FIG. 2. The alignment was generatedusing the Jotun Hein method with the PAM250 residue weight table. Gapsare indicated by dashes; residues that are identical between the twosequences (within 1 distance unit) are boxed.

[0049]FIG. 4 shows the amino acid sequence alignment of SEQ ID NO:3(identified as “264 prot”) and murine NTPase (“mur ntpase”). The aminoacid residues are designated as for FIG. 2. The alignment was generatedas discussed for FIG. 3 Gaps are indicated by dashes; residues that areidentical between the two sequences (within 1 distance unit) are boxed.

[0050]FIG. 5 shows the apyrase conserved regions (ACR) in CD39-L4 inbold. ACR I starts at Phe 53, ACR II starts at Pro 124 and ACR IIIstarts at Met 167. The boxed sections highlight the amino acidsubstitutions that were made in the wild type CD39-L4 amino acidsequence to form mutants designated ACRI, ACRII and ACRIII.

[0051]FIG. 6 (SEQ ID NOS: 6 and 7) shows the nucleotide andcorresponding amino acid sequences of a preferred ACRIII mutantcontaining the following substitutions in the wild type CD39-L4 aminoacid sequence: D168-T, S170-Q and L175-F.

[0052]FIG. 7 shows the ADPase activity of CD39-L4 variants ACRI, ACRIIand ACRIII in comparison to wild type CD39-L4: (1) CD39-L4 ACR I mutant;(2) CD39-L4 ACR II mutant; (3) CD39-L4 ACR III mutant; (4) CD39-L4 wildtype; (5) sCD39; and (6) pSecTag2 vector (Invitrogen).

[0053]FIG. 8 shows the amino acid sequence alignment of SEQ ID NO. 3,SEQ ID NO. 25 (previously identified as SEQ ID NO. 5 in FIG. 5 of U.S.Ser. No. 09/122,449) and human CD39 (“CD39Human.seq”). The alignment wasgenerated using the Jotun Hein method with the PAM250 residue weighttable. Gaps are indicated by dashes; residues that are identical betweenthe two sequences (within 1 distance unit) are boxed.

[0054]FIG. 9 shows the amino acid sequence alignment of SEQ ID NO. 3,SEQ ID NO. 25 (previously identified as SEQ ID NO. 5 in FIG. 6 of U.S.Ser. No. 09/122,449) and the murine NTPase (“mur ntpase”). The alignmentwas generated as discussed for FIG. 8. Gaps are indicated by dashes;residues that are identical between the two sequences (within 1 distanceunit) are boxed.

6. DETAILED DESCRIPTION

[0055] 6.1 Definitions

[0056] The term “nucleotide sequence” refers to a heteropolymer ofnucleotides or the sequence of these nucleotides. The terms “nucleicacid” and “polynucleotide” are also used interchangeably herein to referto a heteropolymer of nucleotides. Generally, nucleic acid segmentsprovided by this invention may be assembled from fragments of the genomeand short oligonucleotide linkers, or from a series of oligonucleotides,to provide a synthetic nucleic acid which is capable of being expressedin a recombinant transcriptional unit comprising regulatory elementsderived from a microbial or viral operon.

[0057] An “oligonucleotide fragment” or a “polynucleotide fragment”,“portion,” or “segment” is a stretch of polypeptide nucleotide residueswhich is long enough to use in polymerase chain reaction (PCR) orvarious hybridization procedures to identify or amplify identical orrelated parts of mRNA or DNA molecules.

[0058] “Oligonucleotides” or “nucleic acid probes” are prepared based onthe cDNA sequence provided in the present invention. Oligonucleotidescomprise portions of the DNA sequence having at least about 15nucleotides and usually at least about 20 nucleotides. Nucleic acidprobes comprise portions of the sequence having fewer nucleotides thanabout 6 kb, usually fewer than about 1 kb. After appropriate testing toeliminate false positives, these probes may be used to determine whethermRNAs are present in a cell or tissue or to isolate similar nucleic acidsequences from chromosomal DNA as described by Walsh, P.S., et al (1992PCR Methods Appl 1:241-250).

[0059] The term “probes” includes naturally occurring or recombinantsingle- or double-stranded nucleic acids or chemically synthesizednucleic acids. They may be labeled by nick translation, Klenow fill-inreaction, PCR or other methods well known in the art. Probes of thepresent invention, their preparation and/or labeling are elaborated inSambrook, J., et al (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, NY; or Ausubel, F. M., et al (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York N.Y., bothincorporated herein by reference.

[0060] The term “stringent” is used to refer to conditions that arecommonly understood in the art as stringent. An exemplary set ofconditions include a temperature of 60-70° C., (preferably about 65° C.)and a salt concentration of 0.70 M to 0.80 M (preferably about 0.75M).Further exemplary conditions include, hybridizing conditions that (I)employ low ionic strength and high temperature for washing, for example,0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employduring hybridization a denaturing agent such as formamide, for example,50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

[0061] The term “recombinant,” as used herein, means that a polypeptideor protein is derived from recombinant (e.g., microbial or mammalian)expression systems. “Microbial” refers to recombinant polypeptides orproteins made in bacterial or fungal (e.g., yeast) expression systems.As a product, “recombinant microbial” defines a polypeptide or proteinessentially free of native endogenous substances and unaccompanied byassociated native glycosylation. Polypeptides or proteins expressed inmost bacterial cultures, e.g., E. coli, will be free of glycosylationmodifications; polypeptides or proteins expressed in yeast will have aglycosylation pattern different from that expressed in mammalian cells.

[0062] The term “recombinant expression vehicle or vector” refers to aplasmid or phage or virus or vector, for expressing a polypeptide from aDNA (RNA) sequence. The expression vehicle can comprise atranscriptional unit comprising an assembly of (1) a genetic element orelements having a regulatory role in gene expression, for example,promoters or enhancers, (2) a structural or coding sequence which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription initiation and termination sequences. Structural unitsintended for use in yeast or eukaryotic expression systems preferablyinclude a leader sequence enabling extracellular secretion of translatedprotein by a host cell. Alternatively, where recombinant protein isexpressed without a leader or transport sequence, it may include anN-terminal methionine residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal product.

[0063] “Recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.The cells can be prokaryotic or eukaryotic. Recombinant expressionsystems as defined herein will express heterologous polypeptides orproteins upon induction of the regulatory elements linked to the DNAsegment or synthetic gene to be expressed.

[0064] The term “open reading frame,” ORF, means a series of tripletscoding for amino acids without any termination codons and is a sequencetranslatable into protein.

[0065] The term “expression modulating fragment,” EMF, means a series ofnucleotide molecules which modulates the expression of an operablylinked ORF or EMF. As used herein, a sequence is said to “modulate theexpression of an operably linked sequence” when the expression of thesequence is altered by the presence of the EMF. EMFs include, but arenot limited to, promoters, and promoter modulating sequences (inducibleelements). One class of EMFs are fragments which induce the expressionof an operably linked ORF in response to a specific regulatory factor orphysiological event.

[0066] As used herein, an “uptake modulating fragment,” UMF, means aseries of nucleotide molecules which mediate the uptake of a linked DNAfragment into a cell. UMFs can be readily identified using known UMFs asa target sequence or target motif with the computer-based systems knownin the art.

[0067] The presence and activity of a UMF can be confirmed by attachingthe suspected UMF to a marker sequence. The resulting nucleic acidmolecule is then incubated with an appropriate host under appropriateconditions and the uptake of the marker sequence is determined. Asdescribed above, a UMF will increase the frequency of uptake of a linkedmarker sequence.

[0068] “Active” refers to those forms of the polypeptide which retainthe biologic and/or immunologic activities of any naturally occurringpolypeptide.

[0069] “Naturally occurring polypeptide” refers to polypeptides producedby cells that have not been genetically engineered and specificallycontemplates various polypeptides arising from post-translationalmodifications of the polypeptide including, but not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidationand acylation.

[0070] “Derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of amino acids suchas ornithine, which do not normally occur in human proteins.

[0071] “Recombinant variant” refers to any polypeptide differing fromnaturally occurring polypeptides by amino acid insertions, deletions,and substitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as cellular trafficking,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology.

[0072] Preferably, amino acid “substitutions” are the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, ora threonine with a serine, i.e., conservative amino acid replacements.“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a polypeptide molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity.

[0073] As used herein, “substantially equivalent” can refer both tonucleotide and amino acid sequences, for example a mutant sequence, thatvaries from a reference sequence by one or more substitutions,deletions, or additions, the net effect of which does not result in anadverse functional dissimilarity between the reference and subjectsequences. Typically, such a mutant sequence varies from one of thoselisted herein by no more than about 20% (i.e., the number ofsubstitutions, additions, and/or deletions in a mutant sequence, ascompared to the corresponding listed sequence, divided by the totalnumber of residues in the mutant sequence is about 0.2 or less). Such amutant sequence is said to have 80% sequence identity to the listedsequence. In one embodiment, a mutant sequence of the invention variesfrom a listed sequence by no more than 10% (90% sequence identity), in avariation of this embodiment, by no more than 5% (95% sequenceidentity), and in a further variation of this embodiment, by no morethan 2% (98% sequence identity). Mutant amino acid sequences accordingto the invention generally have at least 95% sequence identity with alisted amino acid sequence, whereas mutant nucleotide sequence of theinvention can have lower percent sequence identities. For the purposesof the present invention, sequences having substantially equivalentbiological activity and substantially equivalent expressioncharacteristics are considered substantially equivalent. For thepurposes of determining equivalence, truncation of the mature sequenceshould be disregarded.

[0074] Where desired, an expression vector may be designed to contain a“signal or leader sequence” which will direct the polypeptide throughthe membrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

[0075] A polypeptide “fragment,” “portion,” or “segment” is a stretch ofamino acid residues of at least about 5 amino acids, often at leastabout 7 amino acids, typically at least about 9 to 13 amino acids, and,in various embodiments, at least about 17 or more amino acids. To beactive, any polypeptide must have sufficient length to display biologicand/or immunologic activity.

[0076] Alternatively, recombinant variants encoding these same orsimilar polypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polypeptide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

[0077] “Activated” cells as used in this application are those which areengaged in extracellular or intracellular membrane trafficking,including the export of neurosecretory or enzymatic molecules as part ofa normal or disease process.

[0078] The term “purified” as used herein denotes that the indicatednucleic acid or polypeptide is present in the substantial absence ofother biological macromolecules, e.g., polynucleotides, proteins, andthe like. In one embodiment, the polynucleotide or polypeptide ispurified such that it constitutes at least 95% by weight, morepreferably at least 99.8% by weight, of the indicated biologicalmacromolecules present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 1000daltons, can be present).

[0079] The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother component normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

[0080] The term “infection” refers to the introduction of nucleic acidsinto a suitable host cell by use of a virus or viral vector.

[0081] The term “transformation” means introducing DNA into a suitablehost cell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

[0082] The term “transfection” refers to the taking up of an expressionvector by a suitable host cell, whether or not any coding sequences arein fact expressed.

[0083] The term “intermediate fragment” means a nucleic acid between 5and 1000 bases in length, and preferably between 10 and 40 bp in length.

[0084] Each of the above terms is meant to encompasses all that isdescribed for each, unless the context dictates otherwise.

[0085] 6.2 Hybridization Conditions

[0086] Suitable hybridization conditions may be routinely determined byoptimization procedures or pilot studies. Such procedures and studiesare routinely conducted by those skilled in the art to establishprotocols for use in a laboratory. See e.g., Ausubel, et al., CurrentProtocols in Molecular Biology, Vol. 1-2, John Wiley & Sons (1989);Sambrook, et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Vols.1-3, Cold Springs Harbor Press (1989); and Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Cold SpringHarbor, N.Y. (1982), all of which are incorporated by reference herein.For example, conditions such as temperature, concentration ofcomponents, hybridization and washing times, buffer components, andtheir pH and ionic strength may be varied.

[0087] 6.3 Nucleic Acids of the Invention

[0088] The sequences falling within the scope of the present inventionare not limited to the specific sequences herein described, but alsoinclude allelic variations thereof. Allelic variations can be routinelydetermined by comparing the sequence provided in SEQ ID NOs: 1, 2, 24 or26, a representative fragment thereof, or a nucleotide sequence at least99.9% identical to SEQ ID NO: 1, 2, 24 or 26 with a sequence fromanother isolate of the same species. Furthermore, to accommodate codonvariability, the invention includes nucleic acid molecules coding forthe same amino acid sequences as do the specific ORFs disclosed herein.In other words, in the coding region of an ORF, substitution of onecodon for another which encodes the same amino acid is expresslycontemplated.

[0089] Any specific sequence disclosed herein can be readily screenedfor errors by resequencing a particular fragment, such as an ORF, inboth directions (i.e., sequence both strands).

[0090] The present invention further provides recombinant constructscomprising a nucleic acid having the sequence of any one of SEQ ID NO:1, 2, 24 or 26, the mature protein coding sequence or a fragmentthereof. The recombinant constructs of the present invention comprise avector, such as a plasmid or viral vector, into which a nucleic acidhaving the sequence of any one of SEQ ID NO: 1, 2, 24 or 26 or afragment thereof is inserted, in a forward or reverse orientation. Inthe case of a vector comprising one of the ORFs of the presentinvention, the vector may further comprise regulatory sequences,including for example, a promoter, operably linked to the ORF. Forvectors comprising the EMFs and UMFs of the present invention, thevector may further comprise a marker sequence or heterologous ORFoperably linked to the EMF or UMF. Large numbers of suitable vectors andpromoters are known to those of skill in the art and are commerciallyavailable for generating the recombinant constructs of the presentinvention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

[0091] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda PR, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

[0092] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0093] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0094] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0095] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced or derepressed by appropriate means (e.g., temperature shift orchemical induction) and cells are cultured for an additional period.Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

[0096] Included within the scope of the nucleic acid sequences of theinvention are nucleic acid sequences that hybridize under stringentconditions to a fragment of the DNA sequences in FIG. 1, which fragmentis greater than about 10 bp, preferably 20-50 bp, and even greater than100 bp, including 200 bp or greater, 300 bp or greater, 400 bp orgreater, and 500 bp or greater.

[0097] In accordance with the invention, polynucleotide sequences whichencode the novel nucleic acids, or functional equivalents thereof, maybe used to generate recombinant DNA molecules that direct the expressionof that nucleic acid, or a functional equivalent thereof, in appropriatehost cells.

[0098] The nucleic acid sequences of the invention are further directedto sequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. The amino acid sequence variants of the nucleic acids arepreferably constructed by mutating the polynucleotide to give an aminoacid sequence that does not occur in nature. These amino acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite.

[0099] Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells.

[0100] In a preferred method, polynucleotides encoding the novel nucleicacids are changed via site-directed mutagenesis. This method usesoligonucleotide sequences that encode the polynucleotide sequence of thedesired amino acid variant, as well as a sufficient adjacent nucleotideon both sides of the changed amino acid to form a stable duplex oneither side of the site being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:1 83 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982).

[0101] PCR may also be used to create amino acid sequence variants ofthe novel nucleic acids. When small amounts of template DNA are used asstarting material, primer(s) that differs slightly in sequence from thecorresponding region in the template DNA can generate the desired aminoacid variant. PCR amplification results in a population of product DNAfragments that differ from the polynucleotide template encoding thepolypeptide at the position specified by the primer. The product DNAfragments replace the corresponding region in the plasmid and this givesthe desired amino acid variant.

[0102] A further technique for generating amino acid variants is thecassette mutagenesis technique described in Wells et al., Gene 34:315(1985); and other mutagenesis techniques well known in the art, such as,for example, the techniques in Sambrook, et al., supra, and CurrentProtocols in Molecular Biology, Ausubel, et al.

[0103] Due to the inherent degeneracy of the genetic code, other DNAsequences which encode substantially the same or a functionallyequivalent amino acid sequence may be used in the practice of theinvention for the cloning and expression of these novel nucleic acids.Such DNA sequences include those which are capable of hybridizing to theappropriate novel nucleic acid sequence under stringent conditions.

[0104] Furthermore, knowledge of the DNA sequence provided by thepresent invention allows for the modification of cells to permit, orincrease, expression of endogenous CD39-like polypeptides. Cells can bemodified (e.g., by homologous recombination) to provide increasedCD39-like expression by replacing, in whole or in part, the naturallyoccurring CD39-like promoter with all or part of a heterologous promoterso that the cells express CD39-like polypeptides at a higher level. Theheterologous promoter is inserted in such a manner that it isoperatively linked to CD39-like encoding sequences. See, for example,PCT International Publication No. WO94/12650, PCT InternationalPublication No. WO92/20808, and PCT International Publication No.WO91/09955. It is also contemplated that, in addition to heterologouspromoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and themultifunctional CAD gene which encodes carbamyl phosphate synthase,aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may beinserted along with the heterologous promoter DNA. If linked to theCD39-like coding sequence, amplification of the marker DNA by standardselection methods results in co-amplification of the CD39-like codingsequences in the cells.

[0105] The polynucleotides of the present invention also make possiblethe development, through, e.g., homologous recombination or knock outstrategies, of animals that fail to express functional CD39-likepolypeptides or that express a variant of a CD39-like polypeptide. Suchanimals are useful as models for studying the in vivo activities ofCD39-like polypeptides as well as for studying modulators of CD39-likepolypeptides.

[0106] 6.4 Identification of Polymorphisms

[0107] Polymorphisms can be identified in a variety of ways known in theart which all generally involve obtaining a sample from a patient,analyzing DNA from the sample, optionally involving isolation oramplification of the DNA, and identifying the presence of thepolymorphism in the DNA. For example, PCR may be used to amplify anappropriate fragment of genomic DNA which may then be sequenced.Alternatively, the DNA may be subjected to allele-specificoligonucleotide hybridization (in which appropriate oligonucleotides arehybridized to the DNA under conditions permitting detection of a singlebase mismatch) or to a single nucleotide extension assay (in which anoligonucleotide that hybridizes immediately adjacent to the position ofthe polymorphism is extended with one or more labelled nucleotides). Inaddition, traditional restriction fragment length polymorphism analysis(using restriction enzymes that provide differential digestion of thegenomic DNA depending on the presence or absence of the polymorphism)may be performed.

[0108] Alternatively, a polymorphism resulting in a change in the aminoacid sequence could also be detected by detecting a corresponding changein amino acid sequence of the protein, e.g., by an antibody specific tothe variant sequence.

[0109] 6.5 Hosts

[0110] The present invention further provides host cells containing SEQID NO: 1, 2, 24 or 26 of the present invention, wherein the nucleic acidhas been introduced into the host cell using known transformation,transfection or infection methods. The host cell can be a highereukaryotic host cell, such as a mammalian cell, a lower eukaryotic hostcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the recombinant construct intothe host cell can be effected by calcium phosphate transfection, DEAE,dextran mediated transfection, or electroporation (Davis, L., et al.,Basic Methods in Molecular Biology (1986)).

[0111] The host cells containing one of SEQ ID NO: 1, 2, 24 or 26 of thepresent invention, can be used in conventional manners to produce thegene product encoded by the isolated fragment (in the case of an ORF) orcan be used to produce a heterologous protein under the control of theEMF.

[0112] Any host/vector system can be used to express one or more of theORFs of the present invention. These include, but are not limited to,eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9cells, as well as prokaryotic host such as E. coli and B. subtilis. Themost preferred cells are those which do not normally express theparticular polypeptide or protein or which expresses the polypeptide orprotein at low natural level.

[0113] Mature proteins can be expressed in mammalian cells, yeast,bacteria, insect cells or other cells under the control of appropriatepromoters. Cell-free translation systems can also be employed to producesuch proteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

[0114] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

[0115] Recombinant polypeptides and proteins produced in bacterialculture are usually isolated by initial extraction from cell pellets,followed by one or more salting-out, aqueous ion exchange or sizeexclusion chromatography steps. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

[0116] 6.6 Peptides

[0117] The present invention further provides isolated polypeptidesencoded by the nucleic acid fragments of the present invention or bydegenerate variants of the nucleic acid fragments of the presentinvention. Fragments may be fused to carrier molecules such asimmunoglobulins for many purposes, including increasing the valency ofprotein binding sites. For example, fragments of the protein may befused through “linker” sequences to the Fc portion of an immunoglobulin.For a bivalent form of the protein, such a fusion could be to the Fcportion of an IgG molecule. Other immunoglobulin isotypes may also beused to generate such fusions. For example, a protein-IgM fusion wouldgenerate a decavalent form of the protein of the invention. Analogs ofthe polypeptides of the invention can be fused to another moiety ormoieties, e,g., targeting moiety or another therapeutic agent. Suchanalogs may exhibit improved properties such as activity and/orstability. By “degenerate variant” is intended nucleotide fragmentswhich differ from a nucleic acid fragment of the present invention(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of thegenetic code, encode an identical polypeptide sequence. Preferrednucleic acid fragments of the present invention are the ORFs whichencode proteins.

[0118] The invention also provides both full length and mature forms(for example, without a signal sequence or precursor sequence) ofCD39-like polypeptides. The full length form of such proteins isidentified in the sequence listing by translation of the nucleotidesequence of each disclosed clone. The mature form of such protein may beobtained by expression of the full-length polynucleotide in a suitablemammalian cell or other host cell. The sequence of the mature form ofthe protein is also determinable from the amino acid sequence of thefull length form.

[0119] A variety of methodologies known in the art can be utilized toobtain any one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. This isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. In an alternative method, thepolypeptide or protein is purified from bacterial cells which naturallyproduce the polypeptide or protein. One skilled in the art can readilyfollow known methods for isolating polypeptides and proteins in order toobtain one of the isolated polypeptides or proteins of the presentinvention. These include, but are not limited to, immunochromatography,HPLC, size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel, et al., CurrentProtocols in Molecular Biology.

[0120] The polypeptides and proteins of the present invention canalternatively be purified from cells which have been altered to expressthe desired polypeptide or protein. As used herein, a cell is said to bealtered to express a desired polypeptide or protein when the cell,through genetic manipulation, is made to produce a polypeptide orprotein which it normally does not produce or which the cell normallyproduces at a lower level. One skilled in the art can readily adaptprocedures for introducing and expressing either recombinant orsynthetic sequences into eukaryotic or prokaryotic cells in order togenerate a cell which produces one of the polypeptides or proteins ofthe present invention.

[0121] The purified polypeptides are used in in vitro binding assayswhich are well known in the art to identify molecules which bind to thepolypeptides. These molecules include but are not limited to, forexample, small molecules, molecules from combinatorial libraries,antibodies or other proteins. The molecules identified in the bindingassay are then tested for antagonist or agonist activity in in vivotissue culture or animal models that are well known in the art. Inbrief, the molecules are titrated into a plurality of cell cultures oranimals and then tested for either cell/animal death or prolongedsurvival of the animal/cells.

[0122] In addition, the binding molecules may be complexed with toxins,e.g., ricin or cholera, or with other compounds that are toxic to cells.The toxin-binding molecule complex is then targeted to the tumor orother cell by the specificity of the binding molecule for SEQ IDNOs:3-4.

[0123]6.7 Gene Therapy

[0124] Mutations in the CD39-like gene that result in loss of normalfunction of the CD39-like gene product underlie CD39-related humandisease states. The invention comprehends gene therapy to restoreCD39-like activity that would thus be indicated in treating thosedisease states. Delivery of a functional CD39-like gene to appropriatecells is effected ex vivo, in situ, or in vivo by use of vectors, andmore particuarly viral vectors (e.g., adenovirus, adeno-associatedvirus, or a retrovirus), or ex vivo by use of physical DNA transfermethods (e.g., liposomes or chemical treatments). See, for example,Anderson, Nature, supplement to vol. 392, no 6679, pp. 25-30 (1998). Foradditional reviews of gene therapy technology, see Friedmann, Science,244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); andMiller, Nature, 357: 455-460 (1992). Alternatively, it is contemplatedthat in other human disease states, preventing the expression of orinhibiting the activity of CD39-like polypeptides will be useful intreating the disease states. It is contemplated that antisense therapyor gene therapy could be applied to negatively regulate the expressionof CD39-like polypeptides.

[0125] 6.8 Deposit of Clone

[0126] A plasmid containing DNA encoding the ACR III mutant wasdeposited with the American Type Culture Collection (ATCC), 10801University Avenue, Manassas, Va., on Jul. 13, 1999 under the terms ofthe Budapest Treaty (ATCC accession no. PTA-346).

[0127] 6.9 Antibodies

[0128] In general, techniques for preparing polyclonal and monoclonalantibodies as well as hybridomas capable of producing the desiredantibody are well known in the art (Campbell, A. M., MonoclonalAntibodies Technology: Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers, Amsterdam, TheNetherlands (1984); St. Groth, et al., J. Immunol. 35:1-21 (1990);Kohler and Milstein, Nature 256:495-497 (1975)), the trioma technique,the human B-cell hybridoma technique (Kozbor, et al., Immunology Today4:72 (1983); Cole, et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc. (1985), pp. 77-96). In addition, techniques forpreparing chimeric and humanized antibodies (including polypeptidescontaining CDR and/or antigen-binding sequences of antibodies) are wellknown in the art.

[0129] Any animal (mouse, rabbit, etc.) which is known to produceantibodies can be immunized with a peptide or polypeptide of theinvention. Methods for immunization are well known in the art. Suchmethods include subcutaneous or intraperitoneal injection of thepolypeptide. One skilled in the art will recognize that the amount ofthe protein encoded by the ORF of the present invention used forimmunization will vary based on the animal which is immunized, theantigenicity of the peptide and the site of injection.

[0130] The protein which is used as an immunogen may be modified oradministered in an adjuvant in order to increase the protein'santigenicity. Methods of increasing the antigenicity of a protein arewell known in the art and include, but are not limited to, coupling theantigen with a heterologous protein (such as globulin or -galactosidase)or through the inclusion of an adjuvant during immunization.

[0131] For monoclonal antibodies, spleen cells from the immunizedanimals are removed, fused with myeloma cells, such as SP2/0-Ag14myeloma cells, and allowed to become monoclonal antibody producinghybridoma cells.

[0132] Any one of a number of methods well known in the art can be usedto identify the hybridoma cell which produces an antibody with thedesired characteristics. These include screening the hybridomas with anELISA assay, western blot analysis, or radioimmunoassay (Lutz, et al.,Exp. Cell Research. 175:109-124 (1988)).

[0133] Hybridomas secreting the desired antibodies are cloned and theclass and subclass is determined using procedures known in the art(Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniquesin Biochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984)).

[0134] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to proteins of the present invention.

[0135] For polyclonal antibodies, antibody containing antiserum isisolated from the immunized animal and is screened for the presence ofantibodies with the desired specificity using one of the above-describedprocedures.

[0136] The present invention further provides the above-describedantibodies in detectably labeled form. Antibodies can be detectablylabeled through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase,alkaline phosphatase, etc.) fluorescent labels (such as FITC orrhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishingsuch labeling are well-known in the art, for example, see (Sternberger,L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. etal., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129(1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

[0137] The labeled antibodies of the present invention can be used forin vitro, in vivo, and in situ assays to identify cells or tissues inwhich a fragment of the polypeptide of interest is expressed. Theantibodies may also be used directly in therapies or other diagnostics.

[0138] The present invention further provides the above-describedantibodies immobilized on a solid support. Examples of such solidsupports include plastics such as polycarbonate, complex carbohydratessuch as agarose and sepharose, acrylic resins and polyacrylamide andlatex beads. Techniques for coupling antibodies to such solid supportsare well known in the art (Weir, D. M. et al., “Handbook of ExperimentalImmunology” 4th Ed., Blackwell Scientific Publications, Oxford, England,Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press,N.Y. (1974)). The immobilized antibodies of the present invention can beused for in vitro, in vivo, and in situ assays as well as forimmuno-affinity purification of the proteins of the present invention.

[0139] 6.10 Computer Readable Sequences

[0140] In one application of this embodiment, a nucleotide sequence ofthe present invention can be recorded on computer readable media. Asused herein, “computer readable media” refers to any medium which can beread and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention.

[0141] As used herein, “recorded” refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention. A variety ofdata storage structures are available to a skilled artisan for creatinga computer readable medium having recorded thereon a nucleotide sequenceof the present invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable medium. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and Microsoft Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. A skilled artisancan readily adapt any number of dataprocessor structuring formats (e.g.text file or database) in order to obtain computer readable mediumhaving recorded thereon the nucleotide sequence information of thepresent invention.

[0142] By providing the nucleotide sequence of SEQ ID NO: 1, 2, 24 or26, a representative fragment thereof, or a nucleotide sequence at least99.9% identical to SEQ ID NO: 1, 2, 24 or 26 in computer readable form,a skilled artisan can routinely access the sequence information for avariety of purposes. Computer software is publicly available whichallows a skilled artisan to access sequence information provided in acomputer readable medium. Software which implements the BLAST (Altschul,et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag, et al.,Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system maybe used to identify open reading frames (ORFs) within a nucleic acidsequence. Such ORFs may be protein encoding fragments and may be usefulin producing commercially important proteins such as enzymes used infermentation reactions and in the production of commercially usefulmetabolites.

[0143] As used herein, “a computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thenucleotide sequence information of the present invention. The minimumhardware means of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. A skilled artisan can readily appreciate thatany one of the currently available computer-based systems is suitablefor use in the present invention.

[0144] As stated above, the computer-based systems of the presentinvention comprise a data storage means having stored therein anucleotide sequence of the present invention and the necessary hardwaremeans and software means for supporting and implementing a search means.As used herein, “data storage means” refers to memory which can storenucleotide sequence information of the present invention, or a memoryaccess means which can access manufactures having recorded thereon thenucleotide sequence information of the present invention.

[0145] As used herein, “search means” refers to one or more programswhich are implemented on the computer-based system to compare a targetsequence or target structural motif with the sequence information storedwithin the data storage means. Search means are used to identifyfragments or regions of a known sequence which match a particular targetsequence or target motif. A variety of known algorithms are disclosedpublicly and a variety of commercially available software for conductingsearch means are and can be used in the computer-based systems of thepresent invention. Examples of such software includes, but is notlimited to, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). Askilled artisan can readily recognize that any one of the availablealgorithms or implementing software packages for conducting homologysearches can be adapted for use in the present computer-based systems.

[0146] As used herein, a “target sequence” can be any nucleic acid oramino acid sequence of six or more nucleotides or two or more aminoacids. A skilled artisan can readily recognize that the longer a targetsequence is, the less likely a target sequence will be present as arandom occurrence in the database. The most preferred sequence length ofa target sequence is from about 10 to 100 amino acids or from about 30to 300 nucleotide residues. However, it is well recognized that searchesfor commercially important fragments, such as sequence fragmentsinvolved in gene expression and protein processing, may be of shorterlength.

[0147] As used herein, “a target structural motif,” or “target motif,”refers to any rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein targetmotifs include, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

[0148] 6.11 Expression Modulating Sequences

[0149] EMF sequences can be identified within a genome by theirproximity to the ORFs. An intergenic segment, or a fragment of theintergenic segment, from about 10 to 200 nucleotides in length, taken 5′from any ORF will modulate the expression of an operably linked 3′ ORFin a fashion similar to that found with the naturally linked ORFsequence. As used herein, an “intergenic segment” refers to thefragments of a genome which are between two ORF(S) herein described.Alternatively, EMFs can be identified using known EMFs as a targetsequence or target motif in the computer-based systems of the presentinvention.

[0150] The presence and activity of an EMF can be confirmed using an EMFtrap vector. An EMF trap vector contains a cloning site 5′ to a markersequence. A marker sequence encodes an identifiable phenotype, such asantibiotic resistance or a complementing nutrition auxotrophic factor,which can be identified or assayed when the EMF trap vector is placedwithin an appropriate host under appropriate conditions. As describedabove, an EMF will modulate the expression of an operably linked markersequence. A more detailed discussion of various marker sequences isprovided below. A sequence which is suspected of being an EMF is clonedin all three reading frames in one or more restriction sites upstreamfrom the marker sequence in the EMF trap vector. The vector is thentransformed into an appropriate host using known procedures and thephenotype of the transformed host is examined under appropriateconditions. As described above, an EMF will modulate the expression ofan operably linked marker sequence.

[0151] 6.12 Triplex Helix Formation

[0152] In addition, the fragments of the present invention, as broadlydescribed, can be used to control gene expression through triple helixformation or antisense DNA or RNA, both of which methods are based onthe binding of a polynucleotide sequence to DNA or RNA. Polynucleotidessuitable for use in these methods are usually 20 to 40 bases in lengthand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee, et al., Nucl. Acids Res. 6:3073(1979); Cooney, et al., Science 15241:456 (1988); and Dervan, et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)).

[0153] Triple helix-formation optimally results in a shut-off of RNAtranscription from DNA, while antisense RNA hybridization blockstranslation of an mRNA molecule into polypeptide. Both techniques havebeen demonstrated to be effective in model systems. Informationcontained in the sequences of the present invention is necessary for thedesign of an antisense or triple helix oligonucleotide.

[0154] 6.13 Diagnostic Assays and Kits

[0155] The present invention further provides methods to identify theexpression of one of the ORFs of the present invention, or homologthereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention.

[0156] In detail, such methods comprise incubating a test sample withone or more of the antibodies or one or more of nucleic acid probes ofthe present invention and assaying for binding of the nucleic acidprobes or antibodies to components within the test sample.

[0157] Conditions for incubating a nucleic acid probe or antibody with atest sample vary. Incubation conditions depend on the format employed inthe assay, the detection methods employed, and the type and nature ofthe nucleic acid probe or antibody used in the assay. One skilled in theart will recognize that any one of the commonly available hybridization,amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard, T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P, Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

[0158] The test samples of the present invention include cells, proteinor membrane extracts of cells, or biological fluids such as sputum,blood, serum, plasma, or urine. The test sample used in theabove-described method will vary based on the assay formats nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is compatible with the systemutilized.

[0159] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0160] Specifically, the invention provides a compartment kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the probes or antibodies of thepresent invention; and (b) one or more other containers comprising oneor more of the following: wash reagents, reagents capable of detectingpresence of a bound probe or antibody.

[0161] In detail, a compartment kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe.

[0162] Types of detection reagents include labeled nucleic acid probes,labeled secondary antibodies, or in the alternative, if the primaryantibody is labeled, the enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody. One skilled in theart will readily recognize that the disclosed probes and antibodies ofthe present invention can be readily incorporated into one of theestablished kit formats which are well known in the art.

[0163] 6.14 Screening Assays

[0164] Using the isolated proteins of the present invention, the presentinvention further provides methods of obtaining and identifying agentswhich bind to a protein encoded by one of the ORFs from a nucleic acidwith a sequence of one of SEQ ID NO: 1, 2, 24 or 26, or to a nucleicacid with a sequence of one of SEQ ID NO: 1, 2, 24 or 26.

[0165] In detail, said method comprises the steps of: (a) contacting anagent with an isolated protein encoded by one of the ORFs of the presentinvention, or nucleic acid of the invention; and (b) determining whetherthe agent binds to said protein or said nucleic acid.

[0166] The agents screened in the above assay can be, but are notlimited to, peptides, carbohydrates, vitamin derivatives, or otherpharmaceutical agents. The agents can be selected and screened at randomor rationally selected or designed using protein modeling techniques.

[0167] For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention.

[0168] Alternatively, agents may be rationally selected or designed. Asused herein, an agent is said to be “rationally selected or designed”when the agent is chosen based on the configuration of the particularprotein. For example, one skilled in the art can readily adapt currentlyavailable procedures to generate peptides, pharmaceutical agents and thelike capable of binding to a specific peptide sequence in order togenerate rationally designed antipeptide peptides, for example seeHurby, et al., Application of Synthetic Peptides: Antisense Peptides,”In Synthetic Peptides, A User's Guide, W. H. Freeman, NY (1992), pp.289-307, and Kaspczak, et al., Biochemistry 28:9230-8 (1989), orpharmaceutical agents, or the like.

[0169] In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control.

[0170] One class of DNA binding agents are agents which contain baseresidues which hybridize or form a triple helix formation by binding toDNA or RNA. Such agents can be based on the classic phosphodiester,ribonucleic acid backbone, or can be a variety of sulfhydryl orpolymeric derivatives which have base attachment capacity.

[0171] Agents suitable for use in these methods usually contain 20 to 40bases and are designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee, et al., Nucl. AcidsRes. 6:3073 (1979); Cooney, et al., Science 241:456 (1988); and Dervan,et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano,J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).Triple helix-formation optimally results in a shut-off of RNAtranscription from DNA, while antisense RNA hybridization blockstranslation of an mRNA molecule into polypeptide. Both techniques havebeen demonstrated to be effective in model systems. Informationcontained in the sequences of the present invention is necessary for thedesign of an antisense or triple helix oligonucleotide and other DNAbinding agents.

[0172] Agents which bind to a protein encoded by one of the ORFs of thepresent invention can be used as a diagnostic agent, in the control ofbacterial infection by modulating the activity of the protein encoded bythe ORF. Agents which bind to a protein encoded by one of the ORFs ofthe present invention can be formulated using known techniques togenerate a pharmaceutical composition.

[0173] 6.15 Use of Nucleic Acids as Probes

[0174] Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from thenucleotide sequence of the SEQ ID NO: 1, 2, 24 or 26. Because thecorresponding gene is expressed in only one out of 18 tissues tested,namely macrophages, a hybridization probe derived from SEQ ID NO: 1, 2,24 or 26 can be used as an indicator of the presence of macrophage RNAin a sample. Any suitable hybridization technique can be employed, suchas, for example, in situ hybridization.

[0175] PCR as described U.S. Pat. Nos. 4,683,195 and 4,965,188 providesadditional uses for oligonucleotides based upon the nucleotidesequences. Such probes used in PCR may be of recombinant origin, may bechemically synthesized, or a mixture of both. The probe will comprise adiscrete nucleotide sequence for the detection of identical sequences ora degenerate pool of possible sequences for identification of closelyrelated genomic sequences.

[0176] Other means for producing specific hybridization probes fornucleic acids include the cloning of nucleic acid sequences into vectorsfor the production of mRNA probes. Such vectors are known in the art andare commercially available and may be used to synthesize RNA probes invitro by means of the addition of the appropriate RNA polymerase as T7or SP6 RNA polymerase and the appropriate radioactively labelednucleotides.

[0177] The nucleotide sequences may be used to construct hybridizationprobes for mapping their respective genomic sequences. The nucleotidesequence provided herein may be mapped to a chromosome or specificregions of a chromosome using well known genetic and/or chromosomalmapping techniques. These techniques include in situ hybridization,linkage analysis against known chromosomal markers, hybridizationscreening with libraries or flow-sorted chromosomal preparationsspecific to known chromosomes, and the like. The technique offluorescent in situ hybridization of chromosome spreads has beendescribed, among other places, in Verma, et al (1988) Human Chromosomes:A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescentin situ hybridization of chromosomal preparations and other physicalchromosome mapping techniques may be correlated with additional geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981 f). Correlation between the location of anucleic acid on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier or affected individuals.

[0178] The nucleotide sequence may be used to produce purifiedpolypeptides using well known methods of recombinant DNA technology.Among the many publications that teach methods for the expression ofgenes after they have been isolated is Goeddel, (1990) Gene ExpressionTechnology, Methods and Enzymology, Vol 185, Academic Press, San Diego.Polypeptides may be expressed in a variety of host cells, eitherprokaryotic or eukaryotic. Host cells may be from the same species fromwhich a particular polypeptide nucleotide sequence was isolated or froma different species. Advantages of producing polypeptides by recombinantDNA technology include obtaining adequate amounts of the protein forpurification and the availability of simplified purification procedures.

[0179] 6.15.1 Preparation of Sequencing Chips and Arrays

[0180] A basic example is using 6-mers attached to 50 micron surfaces togive a chip with dimensions of 3×3 mm which can be combined to give anarray of 20×20 cm. Another example is using 9-mer oligonucleotidesattached to 10×10 microns surface to create a 9-mer chip, withdimensions of 5×5 mm. 4000 units of such chips maybe used to create a30×30 cm array. In an array in which 4,000 to 16,000 oligochips arearranged into a square array. A plate, or collection of tubes, as alsodepicted, may be packaged with the array as part of the sequencing kit.

[0181] The arrays may be separated physically from each other or byhydrophobic surfaces. One possible way to utilize the hydrophobic stripseparation is to use technology such as the Iso-Grid Microbiology Systemproduced by QA Laboratories, Toronto, Canada.

[0182] Hydrophobic grid membrane filters (HGMF) have been in use inanalytical food microbiology for about a decade where they exhibitunique attractions of extended numerical range and automated counting ofcolonies. One commercially-available grid is ISO-GRIDTM from QALaboratories Ltd. (Toronto, Canada) which consists of a square (60×60cm) of polysulfone polymer (Gelman Tuffryn HT-450, 0.45μ pore size) onwhich is printed a black hydrophobic ink grid consisting of 1600 (40×40)square cells. HGMF have previously been inoculated with bacterialsuspensions by vacuum filtration and incubated on the differential orselective media of choice.

[0183] Because the microbial growth is confined to grid cells of knownposition and size on the membrane, the HGMF functions more like an MPNapparatus than a conventional plate or membrane filter. Peterkin et al.(1987) reported that these HGMFs can be used to propagate and storegenomic libraries when used with a HGMF replicator. One such instrumentreplicates growth from each of the 1600 cells of the ISO-GRID andenables many copies of the master HGMF to be made (Peterkin et al.,1987).

[0184] Sharpe et al. (1989) also used ISO-GRID HGMF form QA Laboratoriesand an automated HGMF counter (MI-100 Interpreter) and RP-100Replicator. They reported a technique for maintaining and screening manymicrobial cultures.

[0185] Peterkin and colleagues later described a method for screeningDNA probes using the hydrophobic grid-membrane filter (Peterkin et al.,1989). These authors reported methods for effective colony hybridizationdirectly on HGMFs. Previously, poor results had been obtained due to thelow DNA binding capacity of the epoxysulfone polymer on which the HGMFsare printed. However, Peterkin et al. (1989) reported that the bindingof DNA to the surface of the membrane was improved by treating thereplicated and incubated HGMF with polyethyleneimine, a polycation,prior to contact with DNA. Although this early work uses cellular DNAattachment, and has a different objective to the present invention, themethodology described may be readily adapted for Format 3 SBH.

[0186] In order to identify useful sequences rapidly, Peterkin etal.(1989) used radiolabeled plasmid DNA from various clones and testedits specificity against the DNA on the prepared HGMFs. In this way, DNAfrom recombinant plasmids was rapidly screened by colony hybridizationagainst 100 organisms on HGMF replicates which can be easily andreproducibly prepared.

[0187] Manipulation with small (2-3 mm) chips, and parallel execution ofthousands of the reactions. The solution of the invention is to keep thechips and the probes in the corresponding arrays. In one example, chipscontaining 250,000 9-mers are synthesized on a silicon wafer in the formof 8×8 mM plates (15 μM/oligonucleotide, Pease et al., 1994) arrayed in8×12 format (96 chips) with a 1 mM groove in between. Probes are addedeither by multichannel pipette or pin array, one probe on one chip. Toscore all 4000 6-mers, 42 chip arrays have to be used, either usingdifferent ones, or by reusing one set of chip arrays several times.

[0188] In the above case, using the earlier nomenclature of theapplication, F=9; P=6; and F+P=15. Chips may have probes of formulaBxNn, where x is a number of specified bases B; and n is a number ofnon-specified bases, so that x=4 to 10 and n=1 to 4. To achieve moreefficient hybridization, and to avoid potential influence of any supportoligonucleotides, the specified bases can be surrounded by unspecifiedbases, thus represented by a formula such as (N)nBx(N)m.

[0189] 6.15.2 Preparation of Support Bound Oligonucleotides

[0190] Oligonucleotides, i.e., small nucleic acid segments, may bereadily prepared by, for example, directly synthesizing theoligonucleotide by chemical means, as is commonly practiced using anautomated oligonucleotide synthesizer.

[0191] Support bound oligonucleotides may be prepared by any of themethods known to those of skill in the art using any suitable supportsuch as glass, polystyrene or Teflon. One strategy is to precisely spotoligonucleotides synthesized by standard synthesizers. Immobilizationcan be achieved using passive adsorption (Inouye & Hondo, 1990); usingUV light (Nagata et al., 1985; Dahlen et al., 1987; Morriey & Collins,1989) or by covalent binding of base modified DNA (Keller et al., 1988;1989); all references being specifically incorporated herein.

[0192] Another strategy that may be employed is the use of the strongbiotin-streptavidin interaction as a linker. For example, Broude et al.(1994) describe the use of Biotinylated probes, although these areduplex probes, that are immobilized on streptavidin-coated magneticbeads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Ofcourse, this same linking chemistry is applicable to coating any surfacewith streptavidin. Biotinylated probes may be purchased from varioussources, such as, e.g., Operon Technologies (Alameda, Calif.).

[0193] Nunc Laboratories (Naperville, Ill.) is also selling suitablematerial that could be used. Nunc Laboratories have developed a methodby which DNA can be covalently bound to the microwell surface termedCovalink NH. CovaLink NH is a polystyrene surface grafted with secondaryamino groups (>NH) that serve as bridge-heads for further covalentcoupling. CovaLink Modules may be purchased from Nunc Laboratories. DNAmolecules may be bound to CovaLink exclusively at the 5′-end by aphosphoramidate bond, allowing immobilization of more than 1 pmol of DNA(Rasmussen et al., 1991).

[0194] The use of CovaLink NH strips for covalent binding of DNAmolecules at the 5′-end has been described (Rasmussen et al., 1991). Inthis technology, a phosphoramidate bond is employed (Chu et al., 1983).This is beneficial as immobilization using only a single covalent bondis preferred. The phosphoramidate bond joins the DNA to the CovaLink NHsecondary amino groups that are positioned at the end of spacer armscovalently grafted onto the polystyrene surface through a 2 nm longspacer arm. To link an oligonucleotide to CovaLink NH via anphosphoramidate bond, the oligonucleotide terminus must have a 5′-endphosphate group. It is, perhaps, even possible for biotin to becovalently bound to CovaLink and then streptavidin used to bind theprobes.

[0195] More specifically, the linkage method includes dissolving DNA inwater (7.5 ng/μl) and denaturing for 10 min. at 95° C. and cooling onice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), isthen added to a final concentration of 10 mM l-MeIm7. A ss DNA solutionis then dispensed into CovaLink NH strips (75 μl/well) standing on ice.

[0196] Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 μl added perwell. The strips are incubated for 5 hours at 50° C. After incubationthe strips are washed using, e.g., Nunc-Immuno Wash; first the wells arewashed 3 times, then they are soaked with washing solution for 5 min.,and finally they are washed 3 times (where in the washing solution is0.4 N NaOH, 0.25% SDS heated to 50° C.).

[0197] It is contemplated that a further suitable method for use withthe present invention is that described in PCT Patent Application WO90/03382 (Southern & Maskos), incorporated herein by reference. Thismethod of preparing an oligonucleotide bound to a support involvesattaching a nucleoside 3′-reagent through the phosphate group by acovalent phosphodiester link to aliphatic hydroxyl groups carried by thesupport. The oligonucleotide is then synthesized on the supportednucleoside and protecting groups removed from the syntheticoligonucleotide chain under standard conditions that do not cleave theoligonucleotide from the support. Suitable reagents include nucleosidephosphoramidite and nucleoside hydrogen phosphorate.

[0198] An on-chip strategy for the preparation of DNA probe for thepreparation of DNA probe arrays may be employed. For example,addressable laser-activated photodeprotection may be employed in thechemical synthesis of oligonucleotides directly on a glass surface, asdescribed by Fodor et al. (1991). incorporated herein by reference.Probes may also be immobilized on nylon supports as described by VanNess et al.(1991); or linked to Teflon using the method of Duncan &Cavalier (1988); all references being specifically incorporated herein.

[0199] To link an oligonucleotide to a nylon support, as described byVan Ness et al. (1991), requires activation of the nylon surface viaalkylation and selective activation of the 5′-amine of oligonucleotideswith cyanuric chloride.

[0200] One particular way to prepare support bound oligonucleotides isto utilize the light-generated synthesis described by Pease et al.,(1994, incorporated herein by reference). These authors used currentphotolithographic techniques to generate arrays of immobilizedoligonucleotide probes (DNA chips). These methods, in which light isused to direct the synthesis of oligonucleotide probes in high-density,miniaturized arrays, utilize photolabile 5′-protectedN-acyl-deoxynucleoside phosphoramidites, surface linker chemistry andversatile combinatorial synthesis strategies. A matrix of 256 spatiallydefined oligonucleotide probes may be generated in this manner and thenused in the advantageous Format 3 sequencing, as described herein.

[0201] 6.15.3 Preparation of Nucleic Acid Fragments

[0202] The nucleic acids to be sequenced may be obtained from anyappropriate source, such as cDNAs, genomic DNA, chromosomal DNA,microdissected chromosome bands, cosmid or YAC inserts, and RNA,including mRNA without any amplification steps. For example, Sambrook etal. (1989) describes three protocols for the isolation of high molecularweight DNA from mammalian cells (p. 9.14-9.23).

[0203] DNA fragments may be prepared as clones in M113, plasmid orlambda vectors and/or prepared directly from genomic DNA or cDNA by PCRor other amplification methods. Samples may be prepared or dispensed inmultiwell plates. About 100-1000 ng of DNA samples may be prepared in2-500 ml of final volume.

[0204] The nucleic acids would then be fragmented by any of the methodsknown to those of skill in the art including, for example, usingrestriction enzymes as described at 9.24-9.28 of Sambrook et al. (1989),shearing by ultrasound and NaOH treatment.

[0205] Low pressure shearing is also appropriate, as described bySchriefer et al. (1990, incorporated herein by reference). In thismethod, DNA samples are passed through a small French pressure cell at avariety of low to intermediate pressures. A lever device allowscontrolled application of low to intermediate pressures to the cell. Theresults of these studies indicate that low-pressure shearing is a usefulalternative to sonic and enzymatic DNA fragmentation methods.

[0206] One particularly suitable way for fragmenting DNA is contemplatedto be that using the two base recognition endonuclease, CviJI, describedby Fitzgerald et al. (1992). These authors described an approach for therapid fragmentation and fractionation of DNA into particular sizes thatthey contemplated to be suitable for shotgun cloning and sequencing. Thepresent inventor envisions that this will also be particularly usefulfor generating random, but relatively small, fragments of DNA for use inthe present sequencing technology.

[0207] The restriction endonuclease CviJI normally cleaves therecognition sequence PuGCPy between the G and C to leave blunt ends.Atypical reaction conditions, which alter the specificity of this enzyme(CviJI**), yield a quasi-random distribution of DNA fragments form thesmall molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992)quantitatively evaluated the randomness of this fragmentation strategy,using a CviJI** digest of pUC 19 that was size fractionated by a rapidgel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed thatCviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, andthat new sequence data is accumulated at a rate consistent with randomfragmentation.

[0208] As reported in the literature, advantages of this approachcompared to sonication and agarose gel fractionation include: smalleramounts of DNA are required (0.2-0.5 ug instead of 2-5 μg); and fewersteps are involved (no preligation, end repair, chemical extraction, oragarose gel electrophoresis and elution are needed). These advantagesare also proposed to be of use when preparing DNA for sequencing byFormat 3.

[0209] Irrespective of the manner in which the nucleic acid fragmentsare obtained or prepared, it is important to denature the DNA to givesingle stranded pieces available for hybridization. This is achieved byincubating the DNA solution for 2-5 minutes at 80-90° C. The solution isthen cooled quickly to 2° C. to prevent renaturation of the DNAfragments before they are contacted with the chip. Phosphate groups mustalso be removed from genomic DNA by methods known in the art.

[0210] 6.15.4 Preparation of DNA Arrays

[0211] Arrays may be prepared by spotting DNA samples on a support suchas a nylon membrane. Spotting may be performed by using arrays of metalpins (the positions of which correspond to an array of wells in amicrotiter plate) to repeated by transfer of about 20 nl of a DNAsolution to a nylon membrane. By offset printing, a density of dotshigher than the density of the wells is achieved. One to 25 dots may beaccommodated in 1 mm², depending on the type of label used. By avoidingspotting in some preselected number of rows and columns, separatesubsets (subarrays) may be formed. Samples in one subarray may be thesame genomic segment of DNA (or the same gene) from differentindividuals, or may be different, overlapped genomic clones. Each of thesubarrays may represent replica spotting of the same samples. In oneexample, a selected gene segment may be amplified from 64 patients. Foreach patient, the amplified gene segment may be in one 96-well plate(all 96 wells containing the same sample). A plate for each of the 64patients is prepared. By using a 96-pin device, all samples may bespotted on one 8×12 cm membrane. Subarrays may contain 64 samples, onefrom each patient. Where the 96 subarrays are identical, the dot spanmay be 1 mm² and there may be a 1 mm space between subarrays.

[0212] Another approach is to use membranes or plates (available fromNUNC, Naperville, Ill.) which may be partitioned by physical spacerse.g. a plastic grid molded over the membrane, the grid being similar tothe sort of membrane applied to the bottom of multiwell plates, orhydrophobic strips. A fixed physical spacer is not preferred for imagingby exposure to flat phosphor-storage screens or x-ray films.

[0213] 6.15.5 Sequence Comparisons

[0214] BLAST, which stands for Basic Local Alignment Search Tool, isused to search for local sequence alignments (Altschul, S. F., (1993) JMol Evol 36:290-300; Altschul, S. F., et al (1990) J Mol Biol215:403-10). BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. Whereas it is ideal for matcheswhich do not contain gaps, it is inappropriate for performingmotif-style searching. The fundamental unit of BLAST algorithm output isthe High-scoring Segment Pair (HSP).

[0215] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0216] 6.16 SEQ ID NOs

[0217] Referring to FIG. 1, SEQ ID NO: 1 is the nucleotide sequence ofan expressed sequence tag corresponding to a polynucleotide isolatedfrom a cDNA library of human fetal liver-spleen. SEQ ID NO:2 is anextended version of SEQ ID NO:1 obtained as described in Example 2, andthe encoded polypeptide in SEQ ID NO: 3 is referred to herein asCD39-L4. SEQ ID NO:2 encodes a polypeptide having the amino acidsequence of SEQ ID NO:3 (shown in FIG. 2). The open reading framecorresponding to SEQ ID NO:3 starts at nucleotide 246, as numbered fromthe 5′ end of SEQ ID NO:2. This open reading frame encodes a polypeptide428 amino acids in length. The estimated molecular weight of theunglycosylated polypeptide is approximately 47.52 kDa.

[0218] Protein database searches with the BLAST algorithm indicate thatSEQ ID NO:3 is homologous to the CD39 family. FIG. 3 shows the aminoacid sequence alignment between SEQ ID NO:3 (identified as “246 prot”)and human CD39 (“CD39Human.seq”), indicating that the two sequencesshare 30% amino acid sequence identity. Moreover, a higher degree ofhomology between the apyrase conserved regions (Kaczmarek et al., J.Biol. Chem. 271:33116-33122 (1996) is observed. In particular, an almostperfect match to a putative ATP-binding region was found from aminoacids 54-58, DAGST (DAGSS in CD39). In addition, the DLGGASTQ motif(DLGGASTQ in CD39), which is very well conserved among ATPDases, isfound from amino acids 199-206 in SEQ ID NO:3. Other regions conservedin apyrases were found from amino acids 129-134, ATAGLR (ATAGMR in CD39)and from amino acids 169-173, GSDEG (GQEEG in CD39).

[0219] SEQ ID NO:3 differs from CD39 in that SEQ ID NO:3 contains ahydrophobic stretch of 22 amino acids at its amino terminus, which isindicative of a leader peptide. SEQ ID NO:3 also lacks the transmembranedomain found at the carboxyl terminus of CD39. These features indicatethat SEQ ID NO:3 is a soluble ATPDase.

[0220] SEQ ID NO:3 shares an even higher degree of homology (86%identity) with a murine NTPase, as shown in the amino acid sequencealignment presented in FIG. 4 (SEQ ID NO:3 is identified as “246 prot,”and mouse CD39 as “mur ntpase”).

[0221] The message encoding SEQ ID NO:3 is tightly regulated in atissue-specific manner. An expression study using a semiquantitativePCR/Southern blot approach revealed a significant level of expression inmacrophage. In contrast, human CD39 is expressed in tissues such asplacenta, lung, skeletal muscle, kidney, and heart.

[0222] SEQ ID NO: 4 is a polynucleotide sequence for CD39-L4 that wasamplified from a macrophage cDNA library. SEQ ID NO: 5 is thecorresponding amino acid sequence (and is identical to SEQ ID NO: 3).

[0223] SEQ ID NO: 6 is the polynucleotide sequence for a CD39-L4 variantdesignated ACRIII, wherein the following amino acid substitutions havebeen made: D168-T, S170-Q and L175-F; SEQ ID NO: 7 is the correspondingamino acid sequence.

[0224] SEQ ID NO: 8 is the genomic sequence for the human CD39-L4 gene;exons appear at nucleotides 1-288 (exon 1), 1281-1580 (exon 2),1820-1855 (exon 3) 2467-2555 (exon 4), 2863-2942 (exon 5), 3889-3950(exon 6), 4894-4995 (exon 7), 5847-5987 (exon 8), 6966-7138 (exon 9) and8556-9365 (exon 10).

[0225] SEQ ID NO: 24 is the polynucleotide sequence for a CD39-L4 splicevariant that creates an isoform designated CD39-L66. SEQ ID NO: 25 isthe corresponding amino acid sequence.

[0226] SEQ ID NO: 26 is the polynucleotide sequence for CD39-L2. SEQ IDNO: 27 is the corresponding amino acid sequence.

[0227] SEQ ID NO: 42 is the complete genomic sequence for the humanCD39-L4 gene; exons appear at nucleotides 245-461, 1454-1533, 2734-2877,4364-4439, 4679-4714, 5326-5414, 5723-5802, 6751-6812, 7758-7859,8712-8852, 9831-9887, 10383-10498, 11916-12002 and 14472-14526.

[0228] 6.17 Uses of Novel CD39-Like Polypeptides and Antibodies

[0229] Polypeptides of the invention having ATPDase, including NDPase,activity are useful for inhibiting platelet function and can thereforebe employed in the prophylaxis or treatment of pathological conditionscaused by or involving thrombosis or excessive coagulation or excessiveplatelet aggregation, such as myocardial infarction, cerebral ischemia,angina, and the like. Polypeptides of the invention can also be used inthe maintenance of vascular grafts. Platelet function can be measured byany of a number of standard assays, such as, for example, the plateletaggregation assay described in Example 5.

[0230] Such pathological conditions include conditions caused by orinvolving arterial thrombosis, such as coronary artery thrombosis andresulting myocardial infarction, cerebral artery thrombosis orintracardiac thrombosis (due to, e.g., atrial fibrillation) andresulting stroke, and other peripheral arterial thrombosis andocclusion; conditions associated with venous thrombosis, such as deepvenous thrombosis and pulmonary embolism; conditions associated withexposure of the patient's blood to a foreign or injured tissue surface,including diseased heart valves, mechanical heart valves, vasculargrafts, and other extracorporeal devices such as intravascular cannulas,vascular access shunts in hemodialysis patients, hemodialysis machinesand cardiopulmonary bypass machines; and conditions associated withcoagulapathies, such as hypercoagulability and disseminatedintravascular coagulopathy. Co-administration of other agents suitablefor treating the pathological condition, e.g., other anti-coagulationagents, is also contemplated.

[0231] CD39-L4 and CD39-L2 are uniquely specific for ADP and do notsubstantially hydrolyze ATP. Thus, adverse side effects from hydrolysisof circulating ATP are avoided.

[0232] For instance, ATP is known to act as an extracellular signal inmany tissues. In the heart, extracellular ATP modulates ionic processesand contractile function (for review see Burnstock, G.,Neuropharmacology 36:1127). Recently, it has been shown thatextracellular ATP markedly inhibits glucose transport in ratcardiomyocytes (Fisher, Y. et al., J. Biol. Chem. 274:755-761. Anothersource of extracellular ATP is that released from parenchymal cellsunder hypoxic or ischemic conditions (Skobel, E., and Kammermeier, H.Biochim. Biophys. Acta 1362:128-134). ATP is also involved in themodulation of anti-IgE-induced release of histamine from human lung mastcells (Schulman, E. S., et al., Am. J. Respir. Cell Mol. Biol.20:520-537).

[0233] CD39-L4 and CD39-L2 polypeptides of the invention are thusexpected to be useful in modulating disease states (including plateletaggregation, inflammation and apoptosis) associated with ADP or otherpurinergic signaling by reducing the levels of NDPs.

[0234] The ability of CD39-L4 and CD39-L2 to hydrolyze NDPs other thanADP has implications outside the circulatory system. For instance, ithas been reported that UDP is the most potent agonist for the human P2Y₆receptor. Communi, et al., Bioch Bioph Res Com 222:303-308 (1996). Thisreceptor is expressed in several tissues including infiltrating T cellspresent in inflammatory bowel disease. Somers, et al., Lab Invest78:1375-1383 (1998). In this microenvironment, a molecule with theenzymatic properties of CD39-L4 (including CD39-L4, the ACR III mutantthereof, CD39-L66, and CD39-L2) could influence (i.e., enhance orinhibit) T cell responses by modifying the extracellular half-life ofUDP. Thus, the invention contemplates use of polypeptides of theinvention for prophylaxis or treatment of inflammation related disordersincluding disorders involving sepsis or systemic inflammatory responsesyndrome or SIRS (and associated conditions such as fever, tachycardia,tachypnea, cytokine overstimulation, increased vascular permeability,hypotension, complement activation, disseminated intravascularcoagulation, anemia, thrombocytopenia, leukopenia, pulmonary edema,adult respiratory distress syndrome, intestinal ischemia, renalinsufficiency and failure, metabolic acidosis and multiorgan dysfunctionsyndrome), including SIRS secondary to surgery, traumatic injury,hemorrhage, bums, endotoxin, cytokine overstimulation; thrombosis;atherosclerosis; acute pancreatitis; dermatosis, including psoriasis;cirrhosis, reperfusion injury; asthma; multiple sclerosis; arthritis,including rheumatoid arthritis, reactive arthritis and chronicinflammatory arthritis; vasculitis; glomerulonephritis; lupus;myasthenia gravis; experimental allergic encephalomyelitis (EAE); otherautoimmune disorders; ulcerative colitis; Crohn's disease; inflammatorybowel disease; necrotizing enterocolitis; pancreatic cell damage fromdiabetes mellitus type 1; hemodialysis; leukapheresis; granulocytetransfusion associated syndrome; rejection reactions after allograft andxenograft transplantation, including graft versus host disease; or otherinflammatory disorders.

[0235] Nucleotides have been reported to be agonists of apoptosis andecto-apyrases may also be used to inhibit apoptosis.

[0236] Another role for CD39-L4 has been suggested by the report thatmouse CD39-L4 maps closely to a locus associated with audiogenic brainseizures in mice. See Chadwick, et al., Genomics 50:357-367 (1998);Seyfried, et al., Genetics 99:117-126 (1981). This locus, known asAsp-1, is thought to be linked or to correspond to a factor thatinfluences Ca²⁺-ATPase activity. Neumann, et al., Behav. Genetics20:307-323 (1990). Thus, CD39-L4 and CD39-L2 may play a role inneurological disorders, particularly since a CD39-L2 variant has beenobserved to be expressed in brain.

[0237] In addition, elevated levels of nucleotides have been associatedwith cancer conditions, including endometrial cancer, myeloid leukemiaand melanoma. Thus, CD39-L4 and CD39-L2 polypeptides of the inventionare expected to be useful in preventing or treating such conditions byreducing circulating or local levels of nucleotide diphosphates.

[0238] Additionally, the polypeptides of the invention can be used asmolecular weight markers, and as a food supplement. A polypeptideconsisting of SEQ ID NO:3, for example, has a molecular mass ofapproximately 47.52 kD in its unglycosylated form. Protein foodsupplements are well known and the formulation of suitable foodsupplements including polypeptides of the invention is within the levelof skill in the food preparation art.

[0239] The polypeptides of the invention are also useful for makingantibody substances that are specifically immunoreactive with CD39-likeproteins. Antibodies and portions thereof (e.g., Fab fragments) whichbind to the polypeptides of the invention can be used to identify thepresence of such polypeptides in a sample. For example, the level of thenative protein corresponding to SEQ ID NO:3 in a blood sample can bedetermined as an indication of vascular condition. Such determinationsare carried out using any suitable immunoassay format, and anypolypeptide of the invention that is specifically bound by the antibodycan be employed as a positive control.

[0240] Additionally, the polypeptides of the invention are useful formodulating the ratios of levels of adenosine molecules in vivo toregulate homeostasis. Adenosine diphosphate (ADP) is an agonist ofplatelet activation and aggregation. It has been demonstrated that theP2Y receptor (and others including P2T and P2Y1 and potentially others)transduces this signal. Adenosine triphosphate (ATP) also binds to thisreceptor, but acts as an antagonist. Therefore, the ratios of levels ofATP/ADP can significantly influence in vivo platelet activation andaggregation. Agents that specifically decrease levels of ADP not onlydecrease the amount of agonist available to signal, but also increasethe relative antagonistic effects of ATP, because of less competitionfor the common receptor.

[0241] CD39-L2 and/or CD39-L4 may be involved in cancer cell generation,proliferation or metastasis. Detection of decreased levels of CD39-L2 orCD39-L4 polynucleotides or polypeptides may be useful for the diagnosisand/or prognosis of one or more types of cancer. For example, thedecreased expression of CD39-L2 or CD39-L4 polynucleotide/polypeptidemay indicate a hereditary risk of cancer, a precancerous condition, oran ongoing malignancy. A defect in the CD39-L2 or CD39-L4 gene may beassociated with a cancer condition. Identification of single nucleotidepolymorphisms associated with cancer or a predisposition to cancer mayalso be useful for diagnosis or prognosis.

[0242] Cancer treatments promote tumor regression by inhibiting tumorcell proliferation, inhibiting angiogenesis (growth of new blood vesselsthat is necessary to support tumor growth) and/or prohibiting metastasisby reducing tumor cell motility or invasiveness. Therapeuticcompositions comprising CD39-L2 and/or CD39-L4 may be effective in adultand pediatric oncology including in endometrial cancer, malignantmelanoma and myeloid leukemia.

[0243] Studies have shown that increased nucleotide phosphates, such asATP and ADP, promote cell cycle progression through activation of P2Y2receptors in endometrial tumor cells (Katzur et al., J. Clin.Endocrinol. Metab. 84: 4085-91, 1999) and less differentiated myeloidleukocytes have enhanced expression of endogenous nucleotides ordecreased expression of ecto-nucleosidases (Clifford et al., Am. J.Physiol. 273: C973-87, 1997). In addition, increased ecto-ATPaseactivity, such as CD-39 activity, causes a decrease in tumor progressiondue to increased immunological recognition. (Dzhandzhugazyan et al.,FEBS Lett. 430: 227-30, 1998). It may be that the ratio of nucleotidediphosphates to nucleotide triphosphates may promote tumor progressionby increasing cellular proliferation and reducing immunologicalrecognition of tumor cells. Therapeutic treatments which alter thesenucleotide ratios, such as CD39-L2 or CD39-L4, may promote tumorregression.

[0244] CD39-L2 or CD39-L4 polypeptides, polynucleotides, or modulators(preferably CD39-L2 or CD39-L4 stimulators of enzymatic activity) may beadministered to treat cancer. Therapeutic compositions can beadministered in therapeutically effective dosages alone or incombination with adjuvant cancer therapy such as surgery, chemotherapy,radiotherapy, thermotherapy, and laser therapy, and may provide abeneficial effect, e.g. reducing tumor size, slowing rate of tumorgrowth, inhibiting metastasis, increasing tumor cell immune recognition,or otherwise improving overall clinical condition, without necessarilyeradicating the cancer.

[0245] The compositions can also be administered in therapeuticallyeffective amounts as a portion of an anti-cancer cocktail. Ananti-cancer cocktail is a mixture of CD39-L2 and/or CD39-L4 polypeptideor modulator with one or more anti-cancer drugs in addition to apharmaceutically acceptable carrier for delivery. The use of anti-cancercocktails as a cancer treatment is routine. Anti-cancer drugs that arewell known in the art and can be used as a treatment in combination withthe polypeptide or modulator of the invention include: Actinomycin D,Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin,Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide,Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin,Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium,Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide,Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a,Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog),Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan,Mercaptopurine, Mesna, Methotrexate (MTX). Mitomycin, Mitoxantrone HCl,Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifencitrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristinesulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2,Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine Sulfate.

[0246] In addition, CD39-L2 and/or CD39-L4 therapeutic compositions maybe used for prophylactic treatment of cancer. There are hereditaryconditions and/or environmental situations (e.g. exposure tocarcinogens) known in the art that predispose an individual todeveloping cancers. Under these circumstances, it may be beneficial totreat these individuals with therapeutically effective doses of CD39-L2and/or CD39-L4 to reduce the risk of developing cancers.

[0247] In vitro models can be used to determine the effective doses ofCD39-L2 and/or CD39-IA as potential cancer treatments. These in vitromodels include proliferation assays of cultured tumor cells, growth ofcultured tumor cells in soft agar (see Freshney, (1987) Culture ofAnimal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch18 and Ch 21), tumor systems in nude mice as described in Giovanella etal., J. Natl. Can. Inst., 52: 921-30 (1974), mobility and invasivepotential of tumor cells in Boyden Chamber assays as described inPilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesisassays such as induction of vascularization of the chick chorioallantoicmembrane or induction of vascular endothelial cell migration asdescribed in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97 (1999) andLi et al., Clin. Exp. Metastasis, 17:423-9 (1999) respectively. Suitabletumor cells lines are available, e.g. from American Type Tissue CultureCollection catalogs.

[0248] The polypeptides of the invention are administered by any routethat delivers an effective dosage to the desired site of action. Thedetermination of a suitable route of administration and an effectivedosage for a particular indication is within the level of skill in theart. For treatment of vascular disease, polypeptides according to theinvention are generally administered intravenously. In vivo murinestudies with soluble human CD39 have shown that mice injectedintravenously with 50 mg recombinant soluble human CD39 in 100 mlsterile saline had biologically active CD39 in their sera for anextended period of time, with an elimination half-life of almost 2 days(Gayle, R. B., et al., J. Clinical Invest. 101: 1851-1859 (1998)).Suitable dosage ranges for the polypeptides of the invention can beextrapolated from these dosages or from similar studies in appropriateanimal models. Dosages can then be adjusted as necessary by theclinician to provide maximal therapeutic benefit.

[0249] 6.18 Pharmaceutical Formulations and Routes of Administration

[0250] A protein of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinantsources) may be administered to a patient in need, by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s) at doses to treat or ameliorate a variety of disorders.Such a composition may also contain (in addition to protein and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2,G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.

[0251] The pharmaceutical composition may further contain other agentswhich either enhance the activity of the protein or compliment itsactivity or use in treatment. For example, CD39-L2 or CD39-L4 may beco-administered with platelet ADP receptor antagonists, e.g. ATPderivatives, ADP derivatives. Such additional factors and/or agents maybe included in the pharmaceutical composition to produce a synergisticeffect with protein of the invention, or to minimize side effects.Conversely, protein of the present invention may be included informulations of the particular cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent to minimize side effects of the cytokine, lymphokine, otherhematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent. A protein of the present invention may beactive in multimers (e.g., heterodimers or homodimers) or complexes withitself or other proteins. As a result, pharmaceutical compositions ofthe invention may comprise a protein of the invention in such multimericor complexed form.

[0252] Techniques for formulation and administration of the compounds ofthe instant application may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

[0253] In practicing the method of treatment or use of the presentinvention, a therapeutically effective amount of protein of the presentinvention is administered to a mammal having a condition to be treated.Protein of the present invention may be administered in accordance withthe method of the invention either alone or in combination with othertherapies such as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors.

[0254] 6.18.1. Routes of Administration

[0255] Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof protein of the present invention used in the pharmaceuticalcomposition or to practice the method of the present invention can becarried out in a variety of conventional ways, such as oral ingestion,inhalation, topical application or cutaneous, subcutaneous,intraperitoneal, parenteral or intravenous injection. Intravenousadministration to the patient is preferred.

[0256] Alternately, one may administer the compound in a local ratherthan systemic manner, for example, via injection of the compounddirectly into a arthritic joints or in fibrotic tissue, often in a depotor sustained release formulation. In order to prevent the scarringprocess frequently occurring as complication of glaucoma surgery, thecompounds may be administered topically, for example, as eye drops.Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with a specific antibody,targeting, for example, arthritic or fibrotic tissue. The liposomes willbe targeted to and taken up selectively by the afflicted tissue.

[0257] 6.18.2. Compositions/Formulations

[0258] Pharmaceutical compositions for use in accordance with thepresent invention thus may be formulated in a conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein of the present invention is administered orally, protein of thepresent invention will be in the form of a tablet, capsule, powder,solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention may additionally contain a solid carriersuch as a gelatin or an adjuvant. The tablet, capsule, and powdercontain from about 5 to 95% protein of the present invention, andpreferably from about 25 to 90% protein of the present invention. Whenadministered in liquid form, a liquid carrier such as water, petroleum,oils of animal or plant origin such as peanut oil, mineral oil, soybeanoil, or sesame oil, or synthetic oils may be added. The liquid form ofthe pharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5 to 90% by weight of protein of the present invention, andpreferably from about 1 to 50% protein of the present invention.

[0259] When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. Forinjection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

[0260] For oral administration, the compounds can be formulated readilyby combining the active compounds with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxyrnethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

[0261] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

[0262] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0263] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0264] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

[0265] A pharmaceutical carrier for the hydrophobic compounds of theinvention is a cosolvent system comprising benzyl alcohol, a nonpolarsurfactant, a water-miscible organic polymer, and an aqueous phase. Thecosolvent system may be the VPD co-solvent system. VPD is a solution of3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80; the fraction size ofpolyethylene glycol may be varied; other biocompatible polymers mayreplace polyethylene glycol, e.g. polyvinyl pyrrolidone; and othersugars or polysaccharides may substitute for dextrose. Alternatively,other delivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

[0266] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols. Many of the proteinase inhibitingcompounds of the invention may be provided as salts withpharmaceutically compatible counterions. Such pharmaceuticallyacceptable base addition salts are those salts which retain thebiological effectiveness and properties of the free acids and which areobtained by reaction with inorganic or organic bases such as sodiumhydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine,monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate,triethanol amine and the like.

[0267] The pharmaceutical composition of the invention may be in theform of a complex of the protein(s) of present invention along withprotein or peptide antigens. The protein and/or peptide antigen willdeliver a stimulatory signal to both B and T lymphocytes. B lymphocyteswill respond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunoglobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

[0268] The amount of protein of the present invention in thepharmaceutical composition of the present invention will depend upon thenature and severity of the condition being treated, and on the nature ofprior treatments which the patient has undergone. Ultimately, theattending physician will decide the amount of protein of the presentinvention with which to treat each individual patient. Initially, theattending physician will administer low doses of protein of the presentinvention and observe the patient's response. Larger doses of protein ofthe present invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about10 mg, more preferably about 0.1 μg to about 1 mg) of protein of thepresent invention per kg body weight. For compositions of the presentinvention which are useful for bone, cartilage, tendon or ligamentregeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein of theinvention which may also optionally be included in the composition asdescribed above, may alternatively or additionally, be administeredsimultaneously or sequentially with the composition in the methods ofthe invention. Preferably for bone and/or cartilage formation, thecomposition would include a matrix capable of delivering theprotein-containing composition to the site of bone and/or cartilagedamage, providing a structure for the developing bone and cartilage andoptimally capable of being resorbed into the body. Such matrices may beformed of materials presently in use for other implanted medicalapplications.

[0269] The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalciumphosphate. The bioceramics may be altered in composition, suchas in calcium-aluminate-phosphate and processing to alter pore size,particle size, particle shape, and biodegradability. Presently preferredis a 50:50 (mole weight) copolymer of lactic acid and glycolic acid inthe form of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

[0270] A preferred family of sequestering agents is cellulosic materialssuch as alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins ofthe invention may be combined with other agents beneficial to thetreatment of the bone and/or cartilage defect, wound, or tissue inquestion. These agents include various growth factors such as epidermalgrowth factor (EGF), platelet derived growth factor (PDGF), transforminggrowth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growthfactor (IGF).

[0271] The therapeutic compositions are also presently valuable forveterinary applications. Particularly domestic animals and thoroughbredhorses, in addition to humans, are desired patients for such treatmentwith proteins of the present invention The dosage regimen of aprotein-containing pharmaceutical composition to be used in tissueregeneration will be determined by the attending physician consideringvarious factors which modify the action of the proteins, e.g., amount oftissue weight desired to be formed, the site of damage, the condition ofthe damaged tissue, the size of a wound, type of damaged tissue (e.g.,bone), the patient's age, sex, and diet, the severity of any infection,time of administration and other clinical factors. The dosage may varywith the type of matrix used in the reconstitution and with inclusion ofother proteins in the pharmaceutical composition. For example, theaddition of other known growth factors, such as IGF I (insulin likegrowth factor I), to the final composition, may also effect the dosage.Progress can be monitored by periodic assessment of tissue/bone growthand/or repair, for example, X-rays, histomorphometric determinations andtetracycline labeling.

[0272] Polynucleotides of the present invention can also be used forgene therapy. Such polynucleotides can be introduced either in vivo orex vivo into cells for expression in a mammalian subject.Polynucleotides of the invention may also be administered by other knownmethods for introduction of nucleic acid into a cell or organism(including, without limitation, in the form of viral vectors or nakedDNA). Cells may also be cultured ex vivo in the presence of proteins ofthe present invention in order to proliferate or to produce a desiredeffect on or activity in such cells. Treated cells can then beintroduced in vivo for therapeutic purposes.

[0273] 6.18.3. Effective Dosage

[0274] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve its intended purpose. Morespecifically, a therapeutically effective amount means an amounteffective to prevent development of or to alleviate the existingsymptoms of the subject being treated. Determination of the effectiveamounts is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Forexample, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture (i.e., the concentration of the test compound whichachieves a half-maximal inhibition of the C-proteinase activity). Suchinformation can be used to more accurately determine useful doses inhumans.

[0275] A therapeutically effective dose refers to that amount of thecompound that results in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD₅₀ and ED₅₀. Compounds whichexhibit high therapeutic indices are preferred. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. See, e.g., Fingl et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1. Dosage amount and interval may be adjustedindividually to provide plasma levels of the active moiety which aresufficient to maintain the C-proteinase inhibiting effects, or minimaleffective concentration (MEC). The MEC will vary for each compound butcan be estimated from in vitro data; for example, the concentrationnecessary to achieve 50-90% inhibition of the C-proteinase using theassays described herein. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.However, HPLC assays or bioassays can be used to determine plasmaconcentrations.

[0276] Dosage intervals can also be determined using MEC value.Compounds should be administered using a regimen which maintains plasmalevels above the MEC for 10-90% of the time, preferably between 30-90%and most preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

[0277] The amount of composition administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

[0278] 6.18.4. Packaging

[0279] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labelled for treatment of an indicatedcondition.

[0280] The present invention is illustrated in the following examples.Upon consideration of the present disclosure, one of skill in the artwill appreciate that many other embodiments and variations may be madein the scope of the present invention. Accordingly, it is intended thatthe broader aspects of the present invention not be limited to thedisclosure of the following examples.

EXAMPLE 1 Isolation of SEQ ID NO:1 from a cDNA Library of Human FetalLiver-Spleen

[0281] A plurality of novel nucleic acids were obtained from a h2HFLS20WcDNA library prepared from human fetal liver-spleen, as described inBonaldo et al., Genome Res. 6:791-806 (1996), using standard PCR,Sequencing by hybridization sequence signature analysis, and Sangersequencing techniques. The inserts of the library were amplified withPCR using primers specific for vector sequences flanking the inserts.These samples were spotted onto nylon membranes and interrogated witholigonucleotide probes to give sequence signatures. The clones wereclustered into groups of similar or identical sequences, and singlerepresentative clones were selected from each group for gel sequencing.The 5′ sequence of the amplified inserts was then deduced using thereverse M13 sequencing primer in a typical Sanger sequencing protocol.PCR products were purified and subjected to fluorescent dye terminatorcycle sequencing. Single-pass gel sequencing was done using a 377Applied Biosystems (ABI) sequencer. One of these inserts was identifiedas a novel sequence not previously obtained from this library and notpreviously reported in public databases. This sequence is shown in FIG.1 as SEQ ID NO:1.

EXAMPLE 2 Isolation of SEQ ID NO:2 and Determination of a NucleotideSequence Encoding a 428-Amino Acid Protein with Sequence Homology toCD39

[0282] The nucleotide sequence shown in FIG. 1, and labeled SEQ ID NO:2,encodes the translated amino acid sequence SEQ ID NO:3, which is shownin FIG. 2. The extended nucleotide sequence was obtained by isolatingcolonies generated from pools of clones from a human macrophage cDNAlibrary (Invitrogen, Cat. # A550-25). Briefly, the macrophage cDNAlibrary was plated on LB/Amp plates (containing 100 mg/ml ampicillin) ata density of about 40,000 colonies/plate. The colonies were lifted ontonitrocellulose filters and hybridized with a radiolabeled probegenerated from the original clone (i.e., SEQ ID NO:1).

[0283] That the identified clones corresponded to SEQ ID NOs:1 and 2 wasconfirmed by using gene-specific primers (5′-GCTACCTCACTTCCTTTGAG-3′[SEQ ID NO: 9] and 5′-CTGGCTGGTGAAGTTTTCCTC-3′ [SEQ ID NO: 10]) in aPCR-based assay. Then PCR using vector- and gene-specific primers wasemployed to amplify the 5′ portion of the cDNA. Nested primers were usedto generate sequence from the amplified product(s). Laser gene™ softwarewas used to edit and “contig” the partial sequences into a full-lengthsequence. As discussed above, the amino acid sequence has strikinghomology to CD39, which is involved in modulating platelet reactivityduring vascular inflammation. Based in part on the observed sequencesimilarity to CD39, the polypeptide encoded by SEQ ID NO: 2 wasdesignated CD39-L4.

EXAMPLE 3

[0284] A. Expression of SEQ ID NOS. 3 and 5 in COS-7 Cells

[0285] COS-7 cells were grown in DMEM (ATCC) and 10% fetal bovine serum(FBS) (Gibco) to 70% confluence. Prior to transfection the media waschanged to DMEM and 0.5% FCS. Cells were transfected with cDNAs for SEQID NOs. 3 and 5 or with pBGal vector by the FuGENE-6 transfectionreagent (Boehringer). In summary, 4 μl of FuGENE-6 was diluted in 100 μlof DMEM and incubated for 5 minutes. Then, this was added to 1 μg of DNAand incubated for 15 minutes before adding it to a 35 mm dish of COS-7cells. The COS-7 cells were incubated at 37° C. with 5% CO₂. After 24hours, media and cell lysates were collected, centrifuged and dialyzedagainst assay buffer (15 mM Tris pH 7.6, 134 mM NaCl, 5 mM glucose, 3 mMCaCl, and MgCl₂. More robust expression can be achieved using theprotocol described in Example 6 below.

[0286] B. Expression Study Using SEQ ID NO:2

[0287] The expression of SEQ ID NO. 2 in various tissues was analyzedusing a semi-quantitative polymerase chain reaction-based technique.Human cDNA libraries were used as sources of expressed genes fromtissues of interest (adult brain, adult heart, adult kidney, adult lymphnode, adult liver, adult lung, adult ovary, adult placenta, adultspleen, adult testis, bone marrow, fetal kidney, fetal liver, fetalliver-spleen, fetal skin, fetal brain, fetal leukocyte and macrophage).Gene-specific primers (5′-GCTACCTCACTTCCTTTGAG-3′ [SEQ ID NO: 9] and5′-GCAGGTCTCCAAGGAAGTACG-3′ [SEQ ID NO: 11]) were used to amplifyportions of the SEQ ID NO:2 sequence from the samples. Amplifiedproducts were separated on an agarose gel, transferred and chemicallylinked to a nylon filter. The filter was then hybridized with aradioactively labeled (α³³P-dCTP) double-stranded probe generated fromthe full-length SEQ ID NO:2 sequence using a Klenow polymerase,random-prime method. The filters were washed (high stringency) and usedto expose a phosphorimaging screen for several hours. Bands indicatedthe presence of cDNA including SEQ ID NO:2 sequences in a specificlibrary, and thus mRNA expression in the corresponding cell type ortissue.

[0288] Of the 18 human tissues tested, macrophage was the only samplethat provided a signal, indicating that expression of SEQ ID NO:2 istightly regulated. In contrast, the CD39 molecule has been found intissues such as placenta, lung, skeletal muscle, kidney and heart.

EXAMPLE 4 Chromosomal Localization of the Gene Corresponding to SEQ IDNOs:1 and 2

[0289] Chromosome mapping technologies allow investigators to link genesto specific regions of chromosomes. Assignment to chromosome 14 wasperformed with the Coriell cell repository monochromosomal panel #2(NIGMS cell repository). This human rodent somatic cell hybrid panelconsists of DNA isolated from 24 hybrid cell cultures retaining 1 humanchromosome each. The panel was screened with gene-specific primers(5′-GCTACCTCACTTCCTTTGAG-3′ [SEQ ID NO: 9] and5′-CTGGCTGGTGAAGTTTTCCTC-3′ [SEQ ID NO: 10]) that generated a sequencetag site (STS). The Genebridge 4 radiation hybrid panel was alsoscreened (Research Genetics), and the results of the PCR screening weresubmitted to the Whitehead/MIT Radiation Hybrid mapping email server athttp://www-genome.wi.mit.edu.

EXAMPLE 5 Platelet Aggregation Assay

[0290] Blood is anticoagulated with 0.1 volume 3.2% sodium citrate.Platelet-rich plasma (PRP) is prepared with an initial whole bloodcentrifugation (200×g, 15 min., 25° C.) and a second centrifugation ofthe PRP (90×g, 10 min.) to eliminate residual erythrocytes andleukocytes. The stock suspension of PRP is maintained at roomtemperature under ⁵% CO₂-air. The platelet aggregation assay uses atwo-sample, four-channel Whole Blood Lumi-Aggregometor, model 560(Chronolog Corp., Havertown, Pa.). PRP containing 1.22×10⁸ platelets ispreincubated with the sample to be tested for inhibition of aggregationfor 10 min. at 37° C. in a siliconized glass cuvette containing astirring bar, followed by stimulation with either ADP (5 mm), collagen(5 mg/ml), or thrombin (0.1 unit/ml). Platelet aggregation is recordedfor at least 10 min. Data are expressed as the percentage of lighttransmission with platelet-poor plasma equal to 100%.

EXAMPLE 6 Expression and Characterization of CD39-L4 as a SolubleApyrase

[0291] The mammalian ectoapyrase CD39 is an integral membrane proteinwith two transmembrane domains (one at each end of the protein)(Maliszewski, C. R. et al., J. Immunol. 153:3574-3583). Thehydrophobicity profiles for the deduced amino acid sequence of otherfamily members, such as CD39L1 and CD39L3, are very similar to CD39(Chadwick, B. P. and Frischauf A. M., Genomics 50:357-367), suggestingthat these proteins also have two membrane spanning domains. However,CD39-L4 does not appear to have a second transmembrane domain at itsC-terminus, suggesting that the N-terminus hydrophobic region could codefor a secretory signal. To test this hypothesis, CD39-L4 was subclonedinto the mammalian expression vector pCDNA3.1 and a 6-Histidine tag wasinserted into the coding sequence.

[0292] The CD39-L4 cDNA sequence was initially isolated from amacrophage cDNA library (Invitrogen). The sense primer(5′-TTAAAGCTTGGGAAAAGAATGGCCACTTC-3′, SEQ ID NO. 20) with a HindIII siteand the antisense primer (5′-AGACTCGAGGTGGCTCAATGGGAGATGCC-3′, SEQ IDNO. 21) with a XhoI site were used to subclone the coding sequences intothe mammalian expression vector pcDNA3.1 (Invitrogen). The nucleotidesequence of the insert is set forth in SEQ ID NO. 4. In order toimmunologically detect the protein, the coding region was furthermodified so that it would include a Gly-Ser-6His epitope tag immediatelyfollowing Arg²⁴. Briefly, two partially overlapping complementaryoligonucleotides (5′-GCGCTGTCTCCCACAGAGGATCGCATCACCATCACCATCACAACCAGCAGACTTGGTT-3′ (SEQ ID. NO. 22) and5′-AACCAAGTCTGCTGGTTGTGATGGTGATGGTGATGCGATCCTCTGTGGGA GACAGCGC-3′ (SEQID NO. 23)) were used on the CD39-L4 pcDNA3.1 template. The primers wereextended in opposite directions around the plasmid using a 12 cycle PCRprogram (95° C., 1 minute; 60° C., 1 minute; 72° C., 15 minutes)(Stratagene). The reaction was treated with DpnI to digest themethylated parental DNA and then transformed into E. coli. Colonies werescreened for the insert.

[0293] A. Expression in COS Cells and Cellular Localization of CD39-L4

[0294] To ascertain whether CD39-L4-6His is secreted, the coding regionof the CD39-L4-6His protein was inserted into the pcDNA3.1 expressionvector and transiently transfected into COS-7 cells. COS-7 cellsobtained from the American Tissue Type Culture Collection were grown inDMEM supplemented with 10% FBS and 100 units/ml penicillin G and 100μg/ml streptomycin sulfate at 37° C. in 10% CO₂. Transfections wereperformed at 75% confluency in 10 cm plates with Fugene-6 (Roche)according to the manufacturers instructions. The cells in 7 mls ofmedium were incubated with 16 μl of Fugene-6 and 8 μg of DNA for 14-18hours. At the end of the transfection the medium was replaced with DMEMmedium containing low serum (1% FBS). The cells were then incubated for24-48 hours prior to harvesting.

[0295] The CD39-L4-6His was concentrated by treating the cell lysatesand medium with Nickel-NTA agarose (Qiagen) followed by SDS/PAGE andimmunoblot analysis with an antibody against the Arg-Gly-Ser-6Hisepitope. Cells were washed twice with PBS containing 0.5 μg/mlleupeptin, 0.7 μg/ml pepstatin and 0.2,ug/ml aprotinin. After a briefsonication and centrifugation step to clear the lysate, the samples werethen incubated with a Nickel-NTA resin at 4° C. for 2-3 hours. Thehistidine-tagged protein complexed to the resin was washed three timeswith PBS before loading onto a 10% SDS/PAGE gel for Western blotanalysis. CD39-L4 was detected in both the cell lysate and the mediumfrom cells transfected with the CD39-L4-6His expression vector, but notfrom control cells. While the predicted molecular weight of CD39-L4-6Hisis 46 kDa, the immunoreactive protein exhibited a mobility by SDS/PAGEcorresponding to a molecular mass of approximately 51 kDa in the mediaand approximately 48 kDa in the cell lysate. The difference in apparentmolecular weight may be due to posttranslational modications of threepotential N-glycosylation sites in the CD39-L4 predicted amino acidsequence.

[0296] B. Secretion of CD39-L4

[0297] Secretion of CD39-L4 was also examined by treatment of thetransfected cells with brefeldin A, an inhibitor of translocation ofsecretory proteins from the endoplasmic reticulum to the Golgiapparatus. Chadwick, et al., Genomics 50:357-367 (1998). Brefeldin A wasdissolved in ethanol and added to the transfected cells 48 hours aftertransfection. Both control and brefeldin A treated cells were washedonce with PBS and incubated for 8 hours in medium with none or varyingdosages of brefeldin A. Increasing dosages of brefeldin A blockedsecretion of CD39-L4-6His and led to massive intracellular accumulation.

[0298] In addition, the secreted 6His tagged protein was isolated frommedia as described above using Nickel-NTA agarose (Qiagen) and subjectedto SDS-PAGE under both reducing and nonreducing conditions. Western blotanalysis with antibody against the Arg-Gly-Ser-6His epitope showed thatunder nonreducing conditions the mobility of the protein was twice asslow as the reduced monomeric form. Thus, the secreted CD39-L4 iscomposed of disulfide-bonded dimers. It is also possible that selectedCD39-L4 is composed of other higher oligomeric forms generated bynoncovalent interaction of dimeric forms.

[0299] C. CD39-L4 Expression in a Stable Human 293 Cell Line

[0300] CD39-L4 was also expressed in human embryonic carcinoma 293 cellsobtained from the American Type Culture Collection as follows. The 293cells were grown in DMEM/F12 media supplemented with 10% FBS and 100units/ml penicillin G and 100 μg/ml streptomycin sulfate at 37° C. in10% CO₂. Transfections were performed at 60% confluency in a 10-cm platewith Fugene-6 (Roche) according to the instructions of the manufacturer.Briefly, the cells in 8 ml of medium were incubated with 16 μl ofFugene-6 and 4,ug of CD39L4-His₆ pcDNA3.1. After 20 hours the cells weretransferred to T-150 flask and the medium was replaced with 293 mediacontaining 800 μg/ml Geneticin (G418, Life Technologies Inc.). The cellswere fed every two days with selection medium for 20 days.

[0301] D. Purification and Molecular Mass of CD39-L4

[0302] Low serum media (1% FBS) from a 293 stable transfected cell lineor transiently transfected COS-7 cells was collected and centrifuged toclear any cell debris. Protease inhibitors (0.5 ug/ml leupeptin, 0.7ug/ml pepstatin and 0.2 ug/ml aprotinin) were added to the media beforepurification through a Nickel-NTA column (Qiagen). The column was washedwith 10-column volumes of PBS buffer and the protein eluted with 0.1 Msodium acetate, pH 4.5 and 0.3 M NaCl. The eluted protein was collectedas fractions and neutralized with TrisHCl, pH 9 added to a finalconcentration of 70 mM. The fractions with most ADP activity were pooledand concentrated further with Microcon concentrators (Millipore).

[0303] This purified CD39L4 protein was subjected to ultracentrifugationin a 5-15% sucrose density gradient and the sedimentation of CD39L4protein was compared with that of marker proteins of known nativemolecular weights to determine its molecular weight. The centrifugationstudies using a sucrose gradient were done in a Backman L8-M (Fullerton,Calif.) ultracentrifuge equipped with a SW 41 rotor. Samples werecentrifuged at 39,000 rpm for 15 h at 4° C. without using the brake. Theprotein was layered on top of a 5-15% (w/v) sucrose density gradient (10ml) in 10 mM Tris pH 7.5, 150 mM NaCl and 5 mM CaCl₂. Individualfractions were then collected from the bottom of the tube and processedfor activity assays. Western blot, coomassie staining and refractiveindex measurements. The mass of CD39L4 was estimated by comparison ofits mobility with those of standard proteins.

[0304] Most of the CD39L4 activity was observed in the 7.25% region ofthe sucrose gradient located between the 55 kDa and 29% Da markers. Thepeak of CD39L4 immunoreactivity also coincided with the peak of activityyielding an estimated molecular mass of approximately 50 kDa. The peaksof activity and immunoreactivity while in close agreement were not fullysymmetrical because there was a small amount of protein sedimenting at ahigher sucrose concentration of between 8-9.5%. A Western blot of thesefractions showed that they were enriched for dimers although not fullydevoid of monomers. Matching the ADPase activities of fraction #17 (9.4%sucrose) and fraction #27 (7.0% sucrose) first, and then comparing therelative amounts of protein by immunoblotting revealed that the amountof monomeric CD39L4 equaled each other in the two fractions.

[0305] Comparable amounts of ADPase activity from monomer and dimerenriched fractions from the sucrose density gradients demonstrated thatthe dimer does not possess any significant level of enzymatic activity.It is possible that the monomer is the active form of the protein andthat the dimer either represents an inactive form or is a by-product ofthe oxidation of the odd-numbered cysteine residues found in CD39L4.CD39L4 appears to be unique in that its monomeric form has higherenzymatic activity than its dimeric form.

[0306] E. CD39-L4 Expression in Insect Cells

[0307] CD39-L4 was expressed in insect cells as follows. The cDNA wascloned into the insect expression vector pIZ/V5-His (Invitrogen).Briefly, the CD39-L4-6His cDNA in the pCDNA 3.1 vector described abovein Example 6 was cut with HindIII and XhoI and the cDNA insert wasinserted in pIZ/V5-His cleaved by HindIII/XhoI. The resulting vector wastransiently transfected into insect High Five cells (Invitrogen). HighFive cells were grown in serum-free High Five media (Invitrogen) at 27°C. Transfections were performed at 50-60% confluency in a 6-cm platewith Insectin-Plus liposomes (Invitrogen) according to Invitrogen'sprotocol. The cells were incubated with 20 μl of Insectin-Plus liposomesand 1 ml of High Five serum-free medium for 15 minutes. The cells werethen incubated with 10 μg of DNA for four hours at room temperature. Atthe end of the transfection, 2 mls of fresh media were added and thecells were incubated for another 48 hours before transferring to 96-wellplates containing 400 μg/ml Zeocin (Invitrogen). After three weeks ofselection the insect cell media was assayed for the secreted protein byWestern blot of a slot blot. The highest overproducing clone wasrescreened by immunoblotting analysis of media separated on a 10%SDS-polyacrylamide gel.

[0308] CD39-L4 was isolated from the insect media by treating withNickel-NTA agarose (Qiagen) and tested for ADPase activity by measuringinorganic phosphate release as described in Example 9 below. Theactivity of the recombinant insect CD39-L4 was comparable to that of themammalian CD39-L4 expressed from COS-7 cells as described in Example 6.However, the insect CD39-L4 appears to be monomeric and undernonreducing conditions migrates with the monomeric form of mammalianCD39-L4.

[0309] The affinity purified mammalian derived protein (see section D)or media samples from High Five cells expressing CD39-L4 were separatedby SDS-polyacrylamide gel (10%) electrophoresis under reducing (143 mMbeta-mercaptoethanol) or non-reducing conditions. The proteins weretransferred onto the Immobilon-P (Millipore) membrane and incubated witha CD39L4 specific antibody prepared as described in Example 18A below(1:2000) and with a horseradish peroxidase-conjugated anti-mouseantibody (Pierce). The bands were visualized using a chemiluminescentreagent (ECL, Amershan Pharmacia).

[0310] Although the predicted molecular mass is 46 kDa, the mammalianprotein (see section D) was expressed as a major species with a mobilityof around 51 kDa. This shift in mobility is most likely due toglycosylation, as incubation of the protein with peptide N-glycosidase F(PNGase F) results in a mobility shift to around 46 kDa. Interestingly,under non-reducing conditions a second species was also detected with amobility of around 110 kDa when fully glycosylated and around 100 kDawhen deglycosylated. These higher molecular weight species couldcorrespond to dimers held together via a disulfide linkage.

[0311] When protein was expressed in High Five insect cells, therecombination protein was detected as a major band of approximately 46kDa in the culture medium, indicating that CD39L4 is efficientlysecreted. The observed molecular mass of recombinant CD39L4 was in veryclose agreement to that of the deglycosylated mammalian protein. A minorband was also detected at around 100 kDa which could correspond todimers. The recombinant protein was also treated with PNGase F but nochange in mobility was observed, demonstrating that the secreted CD39L4protein is not glycosylated to the same extent as that of the proteinsecreted from 293 cells. Reduction of the disulfide bonds resulted inthe disappearance of the 100 kDa species. The underglycosylatedrecombinant protein isolated from insect cells also demonstratedcomparable relative activity to the fully glycosylated mammalianprotein.

EXAMPLE 7 Assay for ATPase Activity

[0312] Apyrase activity was determined by measuring the amount of[³³P]P_(i) released from [γ³³P]ATP. In summary, 50 μl of samples wereincubated in the presence of 10 μCi of [γ³³P]ATP for one hour at 37° C.The [³³P]P_(t) released and the [γ³³P]ATP were separated by thin layerchromatography (TLC) plates (EM Science). The solvent system consistedof 1 M KH₂PO₄. The separated compounds were scanned for radioactivitywith a Phosphoimager (Molecular Dynamics, Sunnyvale, Calif.) andquantitated by ImageQuant software. COS-7 cells transfected with SEQ IDNOs. 3 and 25 had at least a four fold increase in activity over cellstransfected with the vector alone. Although ATPase activity was present,Example 13 demonstrates that CD39-L4 has significantly more NDPaseactivity.

EXAMPLE 8 Site-Directed Mutagenesis of CD39L4

[0313] Site directed mutagenesis was employed to increase the enzymaticactivity of CD39L4. Amino acid sequence comparisons between CD39 familymembers reveal four highly homologous regions in all five human members(Chadwick and Frischauf, Genomics 50:357-367, 1998). These regions,termed apyrase-conserved regions (ACRs), are present not only in theCD39 family members but other apyrases from species as distant as yeastand plants. Examination of similarities and differences in the CD39 ACRsled to the design of three CD39L4 mutants (see FIG. 5). In thesemutants, codons encoding CD39 ACR specific residues were used to replacecodons from the CD39L4 wild type ACR sequence. Only residues withsignificantly different structural or chemical properties were replaced.A PCR based approach was used to produce these mutations.

[0314] Briefly, the expression vector pCDNA3.1 (Invitrogen) containingthe full coding sequence of the CD39L4 gene (with a 6 Histidine taginserted after Arg 24 in the coding sequence to allow purification ofthe secreted mature form of the protein) was subjected to a PCR-basedsite-directed mutagenesis approach using overlapping oligonucleotides[CD39-L4 ACR I mutant (nt 177-148 and 160-204): 5′-GTG AGT GCT CCC TGCATC TAA CAT AAT TCC-3′ (SEQ ID NO: 12) and 5′-GAT GCA GGG AGC ACT CACACT AGT ATT CAT GTT TAC ACC TTT GTG-3′ (SEQ ID NO: 13); CD39-L4 ACR IImutant (nt 402-359 and 385-415): 5′-GCG TAG TCC TGC TGT TGC CCC TAG GTACAC TGG GGT CTT TTT CC-3′ (SEQ ID NO: 14) and 5′-GCA ACA GCA GGA CTA CGCTTA CTG CCA GAA C-3′ (SEQ ID NO: 15); and CD39-L4 ACR III mutant (nt532-485 and 513-540): 5′-CCC AAG CGA ATA TGC CTT CGT CTT GTC CAG TCA TGATGC TAA CAC TGC-3′ (SEQ ID NO: 16) and 5′-CGA AGG CAT ATT CGC TTG GGTTAC TGT G-3′ (SEQ ID NO: 17)]. After amplification of the whole plasmidwith Pfu DNA polymerase (Stratagene) (95° C./1 min; 60° C./1 min; 72°C./15 min for 12 cycles), the methylated parental DNA was digested withthe restriction enzyme DpnI, leaving only the unmethylated PCR amplifiedproducts. The resulting annealed double-stranded nicked products werethen transformed into bacteria and the resulting colonies were screenedfor the desired mutations by sequencing. The subsequent constructs werefully sequenced to verify that the mutations were in fact introduced andthat no extraneous mutations were generated.

EXAMPLE 9 ACR III Mutant Increases ADPase Activity

[0315] Plasmids containing the mutated and wild type forms of the CD39L4gene were transfected into COS-7 cells. After two days, protein waspurified from the culture medium using a Nickel-NTA resin approach toconcentrate the tagged proteins. These proteins were then assayed forATPase and ADPase activity by measuring the inorganic phosphate released(Wang, T. F., et al., J. Biol. Chem. 273:24814-24821, 1998). Theproteins were incubated in apyrase buffer (15 mM Tris pH 7.4, 135 mMNaCl, 2 mM EGTA and 10 mM glucose) for 1 hour at 37° C. with or without2 mM CaCl₂ or 2 mM MgCl₂. Phosphatase reactions were initiated by theaddition of ADP or ATP to a final concentration of 1 mM. The reaction ofinorganic phosphorus with ammonium molybdate in the presence of sulfuricacid, produces an unreduced phosphomolybdate complex. The absorbance ofthis complex at 340 nm is directly proportional to the inorganicphosphorus concentration (Daly, J. A., and Ertingshausen G., Clin. Chem.18:263 (1972) (Sigma Diagnostics)).

[0316] As seen in FIG. 7, mutations in ACR I and II eliminate activity,whereas the mutations in ACR III increase activity six-fold over wildtype. The increased ADPase activity over wild type is due to acorresponding increase in the amount of protein and not to an increasein the specific activity of the enzyme. Western blots using antibodyagainst the Arg-Gly-Ser-6His epitope of 6His ACRIII compared to 6Hiswildtype CD39-L4 isolated on Nickel-NTA showed that recombinantexpression of ACRIII in COS-7 cells was approximately 6-fold higher thanexpression of the wildtype protein. Therefore, the mutant proteinappears to be synthesized at a higher level than the wild type proteinin COS-7 cells. The replacement of three amino acids in the III region(amino acids 167 to 181 in CD39-L4) and the resulting increase in ADPaseactivity or expression predicts that replacement of additional aminoacids within this region by amino acids from the equivalent region ofCD39 may also enhance the activity or expression of the protein overwild type CD39L4. The increase in ADPase activity or expression overwild type may also be due to the replacement of only one or two of thethree amino acids; this can be confirmed by replacing one or two aminoacids at a time. In addition, changing the nucleotide sequence by makingsilent codon changes at the same position without affecting the aminoacid sequence may also result in increased expression or enzymeactivity.

[0317] The polynucleotide and amino acid sequences of a CD39-L4 varianttermed ACRIII and having the amino acid substitutions D168-T, S170-Q andL175-F compared to wild type CD39-L4 (SEQ ID NO: 5) are set forth in SEQID NOs: 6 and 7, respectively, and in FIG. 6.

EXAMPLE 10 ACR III Mutant and Wild Type Forms are Specific for ADP andnot ATP

[0318] Both the CD39L4 wild type and the CD39L4 variant with mutationsin the ACRIII region hydrolyze ADP. However, when ATP was tested as asubstrate, neither the CD39L4 nor the CD39L4 mutant, ACR III, catalyzedhydrolysis. In contrast, CD39 as a membrane bound molecule (Marcus, etal., The Journal of Clinical Investigation, 99: 1351-1360) or as agenetically engineered soluble form (Gayle, et al., The Journal ofClinical Investigation, 101:1851-1858, 1998) is able to hydrolyze bothATP and ADP substrates efficiently. The specificity that both CD39L4wild type and the CD39L4 ACR III mutant have for ADP is an advantageousfeature that makes these CD39L4-type molecules better antiplatelettherapeutic candidates than CD39, as ADP is the agonist that causesplatelet aggregation. Therapeutics that have both ADPase and ATPaseactivities potentially could create adverse side effects by interferingwith levels of ATP in the circulation.

EXAMPLE 11 Organization of the Human CD39-L4 Gene

[0319] A human CITB BAC genomic library (Research Genetics) was screenedwith gene specific primers [246-16 (nt 5522-5543),5′-CTTCCTTCACTGGGAATTCAGG-3′ (SEQ ID NO: 18) and 246-K4 (nt 4922-4945),5′-CTGTTTACCGAGATGGTTGGAAGC-3′ (SEQ ID NO: 19)] using a PCR based assay.

[0320] Briefly, gene specific primers were used to screen pools of BACDNAs. BAC pools that produced an amplified DNA fragment of the predictedsize were pursued until an individual BAC was identified. BAC63-I18 wasisolated and the CD39-L4 gene sequenced mainly by subcloning PCRamplified regions with gene-specific primers and intron-specificprimers, and to a lesser extent by direct sequencing of BAC DNA. Thepartial sequence is set forth in SEQ ID NO: 8. The CD39-L4 codingsequence was found to be distributed over 14 exons spanning 15 kb ofgenomic DNA as set out in SEQ ID NO: 42; exons appear at nucleotides245-461, 1454-1533, 2734-2877, 4364-4439, 4679-4714, 5326-5414,5723-5802, 6751-6812, 7758-7859, 8712-8852, 9831-9887, 10383-10498,11916-12002 and 14472-14526. The last two exons are differentiallyspliced, producing either CD39-L4 or CD39-L66.

EXAMPLE 12 CD39-L4 and CD39-L2 are Stimulated by Divalent Cations

[0321] The high degree of conservation in the apyrase conserved regionsof CD39-L4 and CD39-L2 suggests similar function to other apyrases. Totest this hypothesis, COS-7 cells were transfected with the CD39-L4-6Hisand CD39-L2myc-His construct as described herein. The medium fromtransfected cells was incubated with Nickel-NTA resin (Qiagen) in orderto capture the 6His tagged protein, the resin was washed with assaybuffer (buffer A: 15 mM Tris pH 7.5, 134 mM NaCl and 5 mM glucose) andthe protein still tethered to the resin in a suspension was assayed forADPase activity. Nucleotidase activity was determined by measuring theamount of inorganic phosphate released from nucleotide substrates usingthe technique of Dlay and Ertingshausen, Clin. Chem. 18:263-265 (1972).In this reaction the complex of inorganic phosphorus with phosphorreagent (ammonium molybdate in the presence of sulfuric acid) producesan unreduced phosphomolybdate compound. The absorbance of this complexat 340 nm is directly proportional to the inorganic phosphorusconcentration. The protein still tethered to the resin as a 30% (50% forCD39-L2) suspension in buffer A was assayed by the addition of thenucleotide to a final concentration of 1 mM and incubated at 37° C. for30 minutes. The reaction was stopped by adding 100 volumes of phosphorreagent. The amount of phosphate released from the reaction wasquantified using a calcium/phosphorus combined standard (Sigma). Theamount of protein used in the assays was estimated by comparing theintensity of the bands in Western blots with that of a series ofstandards of known quantity. CD39-L4 protein from transfected cellsdisplayed a 2.3 fold increase in activity over the cells transfectedwith the vector alone. When Ca²⁺ and Mg²⁺ were added, the activityincreased 3.6 fold and 6 fold, respectively. CD39-L2 protein fromtransfected cells displayed an 8.7 fold increase in activity over thecells transfected with the vector alone. When Ca²⁺ and Mg²⁺ were added,the activity of the CD39-L2 cells increased another 2-3 fold.

EXAMPLE 13 Characterization of CD39-L4 Activity

[0322] CD39-L4 protein was assayed for ADPase activity in the presenceof different kinds of inhibitors of ADPases. Control ecto-apyraseactivity was determined with protein tethered to the Nickel-NTA resin.Both assays were performed as described above except the protein was inbuffer A containing 2 mM CaCl₂ and 2 mM MgCl₂. As shown by Table 1below, inhibitors of phosphatases (F⁻) and adenylate kinase (Ap5A) didnot inhibit activity. The inhibitors of vacuolar ATPases (NEM),mitochondrial ATPases (N3⁻) and Na⁺, K⁻, ATPase (ouabain) did notsignificantly inhibit the Ca²⁺ and Mg²⁺ stimulated activity. However,metal chelators (EDTA and EGTA) significantly inhibited activity. Theseresults show that the overwhelming majority of the activity in theassays originates from a protein bound to the resin with characteristicsof an E-type apyrase. TABLE 1 Inhibition of CD39-L4 activity INHIBITORS% OF CONTROL Control 100 ± 7 Ouabain (1 mM)  96 ± 6 NEM (10 mM) 106 ± 5N3 (1 mM)  100 ± 12 F (10 mM) 113 ± 5 Ap5A (10 μM) 121 ± 9 EGTA (2 mM) 35 ± 3 EDTA (2 mM)  52 ± 3

[0323] As shown in Table 2 below, the nucleotide specificity of CD39-L4was also assayed as described above. The CD39-L4 activity was determinedwith protein tethered to the Ni-NTA resin. The protein was in buffer Acontaining 1 mM EGTA, as well as 2 mM CaCl₂ and MgCl₂. The assay wasstarted by adding the nucleotides to a final concentration of 1 mM. Thevalues below are expressed relative to ADP. The relative activity of thenucleotide triphosphates varies almost seven-fold with ATP being thepoorest substrate. No phosphate release was detected with AMP and ADPwas hydrolyzed at a rate approximately twenty-fold higher than ATP. Theother nucleotide diphosphates (GDP and UDP) were also very efficientlyhydrolyzed by CD39-L4. These results indicate that CD39-L4 defines a newclass of E-type apyrase in humans with a specificity for NDPs asenzymatic substrates. TABLE 2 Substrate specificity of CD39-L4NUCLEOTIDE % OF CONTROL ADP 100 ± 15 ATP  5 ± 1 AMP 0 CTP 26 ± 2 GTP 34± 1 UTP 12 ± 4 CDP 268 ± 11 GDP 334 ± 38 UDP 408 ± 14

[0324] B. Determination of Kinetic Characteristics of CD39L4 ADPaseHydrolysis

[0325] The determination of kinetic parameters for ADP hydrolysis wascarried out in buffer A in the presence of 15 mM CaCl₂, 1 mM ouabain, 10mM NEM, 10 μM Ap5A and concentrations of ADP varying from 0.75 mM to 18mM, at 37° C. for 15 min. The reaction was stopped by adding 100 volumesof phosphorus reagent (Sigma). The amount of phosphate released fromeach reaction was quantitated by comparing the absorbance at 340 nm withthat of a Calcium/Phosphorus standard (Sigma).

[0326] The rate of product release was found to be linear within thefirst 20 minutes of reaction, therefore the initial velocity V_(o) wastaken to be the rate of reaction over the first 15 minutes. V_(o) wasdetermined over a range of ADP concentrations, and each data pointrepresents an average of 3 separate experiments. Curve-fitting of theMichaelis-Menton Equation {V_(o)=(V_(max)[S])/(K_(m)+[S])} to the datapoints was performed by DeltaGraph® 4.0 software (SPSS Inc., Chicago,Ill.) resulted in an R² value equal to 0.994. The V_(max)=1191 pmol/minand K_(m)=12.7 mM were also calculated.

[0327] The amount of CD39L4 protein in each reaction was found to be 60ng and by assuming a molecular weight of 46000 g/mol for CD39L4 protein,the turnover number, k_(cat) of CD39L4 with ADP as a substrate wasdetermined to be 913/min.

[0328] The kinetic data for CD39L4 shows a K_(m) value in the millimolarrange for ADP. However, levels of ADP in the circulation appear to be inthe low micromolar range. This suggests that CD39L4 would be effectivein quenching a sudden rise in the levels of ADP in situations such asplatelet activation, wherein the intracellular levels of ADP could besecreted rapidly to levels optimal for CD39-L4 hydrolysis. Densegranules inside platelets have been shown to contain high concentrationsof ADP, estimated to be up to 0.5 M. Interestingly, as described inExample 17 below, CD39-L2 also exhibits a similar K, value indicating apotential overlapping function for these two enzymes.

EXAMPLE 14 Glycosylation is not Essential for CD39-L4 Activity

[0329] The cDNA encoding CD39L4 predicts three potential N-glycosylationsites. Post-translational modifications such as N-linked glycosylationare common in secreted and membrane-bound mammalian proteins. Thesemodifications may be important for correct protein folding or enzymaticactivity and are not easily reproduced when the proteins are expressedin other organisms such as bacteria. In order to test whether CD39-L4 isglycosylated, COS-7 cells, transfected as described in Example 6, weretreated with tulicamycin (Sigma), which blocks the formation ofN-glycosidic linkages.

[0330] COS-7 cells were grown to 75% confluency and transfected with theCD39-L4-6His construct. After 24 hours, a fraction of the COS-7 cellswere treated with Tunicamycin at a concentration of 5 μg/ml. The mediawas replaced again after 24 hours with fresh tunicamycin and harvestedafter 48 hours. The CD39-L4-6His protein was concentrated by treatingthe media with Nickel-NTA agarose (Qiagen). The resin was washed withassay buffer and the protein still tethered to the resin in a suspensionwas assayed for a shift in electrophoretic nobility as well as itsADPase activity.

[0331] Western blot analysis using an antibody against the 6-His epitoperevealed that the glycosylated CD39-L4 protein isolated from the controlcells had an approximate size of 51 kDa. However, tunicamycin treatedcells had a molecular weight of approximately 46 kDa indicating that theprotein was deglycosylated.

[0332] ADPase activity of the tunicamycin treated cells was assayed asdescribed in Example 12 above. The deglycosylated CD39-L4 protein hadADPase activity comparable to an equal amount of the glycosylatedprotein isolated from control cells. This demonstrates thatglycosylation of the protein is not important for ADPase activity.

[0333] As described in Example 6E above, treatment of the recombinantprotein with peptide N-glycosidase F also resulted in a shift inelectrophoretic mobility, confirming glycosylation of the protein.

[0334] As shown here and in Example 6, neither dimer formation norglycosylation appeared to contribute significantly to the ADPaseactivity of CD39L4, indicating that expression of an active recombinantCD39-L4 product in E. coli is possible.

EXAMPLE 15

[0335] A. Cloning and Expression of CD39-L2

[0336] The CD39-L2 coding sequence (SEQ ID NO: 26) was subcloned intopcDNA3.1/myc-His(+)A (Invitrogen) via the EcoRI and XbaI sites. Briefly,a human adult heart cDNA library (Gibco BRL) was subjected to polymerasechain reaction (PCR) using gene-specific primers L2-5′B(5′-CGTATCCCGCGGGTGGAGGCCGGGGTG-3′, SEQ ID NO: 28) and L2-3′B(5′-CTTCTGCAAGTCCCAGAGCCAGTGTGC-3′, SEQ ID NO: 29). The resultingproducts were diluted 100-fold and subjected to a second round of PCRwith primers L2-5′A (5′-GGAGCCCAAAAGACCGGCTGC-3′, SEQ ID NO: 30) andL2-3′A (5′-TGAAGTCACGTCCAGGACAGG-3′, SEQ ID NO: 31). The productrepresented a single band by agarose gel and was purified and sequencedto confirm its identity. Primers corresponding to the translationalstart region and the carboxy terminal region, excluding the stop codon,of the CD39-L2 coding sequence, L2EcoMet(5′-CGGAATTCAACATGAAAAAAGGTAATCCGTTATGAA-3′, SEQ ID NO: 32) and L2Xba3′(5′-TGTCTAGATGAGGCTGGACTCTTCTG-3′, SEQ ID NO: 33) were used on thepurified DNA to produce a DNA fragment corresponding to the entirecoding region of the CD39-L2 gene, flanked by EcoRI and XbaI sites. ThisPCR product was digested to generate overhang ends that were ligatedinto the EcoRI and XbaI sites of pcDNA3.1/myc-His(+)A. The resultingplasmid allowed expression of the CD39-L2 coding sequence fused in framewith the myc-6His epitope at the carboxy terminus.

[0337] Transfection of COS-7 cells was performed as described below.COS-7 cells obtained from the American Tissue Type Culture Collectionwere grown in DMEM supplemented with 10% FBS and 100 units/ml penicillinG and 100 μg/ml streptomycin sulfate at 37° C. in 10% CO₂. Transfectionswere performed at 75% confluency in 10 cm plates with Fugene-6 accordingto the manufacturer's instructions. The cells in 10 ml of medium wereincubated with 16 μl of Fugene-6 and 4 μg of DNA for 48 hours. Themedium was then replaced by DMEM containing low serum (1% FBS) orserum-free DMEM and incubated for 48 hours before harvesting.

[0338] After collecting the conditioned media from the transfected COScells, cells were washed twice with PBS and then scraped from plates.Upon centrifugation the cells were resuspended in PBS containing 0.5μg/ml leupeptin, 0.7 μg/mL pepstatin and 0.2 μg/mL aprotinin. After abrief sonication, the cytosolic fraction was separated from theinsoluble membrane fraction by centrifugation and protein purified fromthe cytosolic fraction generally as described above for CD39-L4. Forpurification of proteins from the conditioned media, the media wascentrifuged initially to clear any cell debris, adjusted to contain 6 mMsodium phosphate, pH 7.6, 0.5 μg/mL leupeptin, 0.7 μg/mL pepstatin and0.2 μg/mL aprotinin, and incubated at 4 C for 2-3 hours with 100 μL ofNi-NTA resin/10 mL of medium. The Ni-NTA resin was washed twice withTris wash buffer (50 mM Tris HCI, pH 7.5, 300 mM NaCl and 5 mMimidazole), followed by 3 washes with apyrase assay buffer (15 mM TrisHCI, pH 7.5, 134 mM NaCl and 5 mM glucose) and resuspended in a 50%suspension in the same buffer.

[0339] B. Tissue-Specific Expression of CD39-L2

[0340] The expression of CD39-L2 in various tissues was analyzed usingboth a semi-quantitative polymerase chain reaction and a Northern blotanalysis. For the PCR-based analysis, human cDNA libraries (brain,heart, kidney, lung, spleen, testis, fetal brain [SUPERSCRIPT™ cDNAlibraries from Gibco BRL]; lymph node, placenta, bone marrow, leukocyte,stimulated leukocyte [MATCHMAKER cDNA libraries from CLONTECH Labs, PaloAlto, Calif.]; and ovary, fetal liver, macrophage [Invitrogen]) wereused as sources of expressed genes from tissues of interest. Plasmid DNA(20 ng) from each library was used as template for PCR amplification.CD39-L2 gene-specific primers (5′-CGTATCCCGCGGGTGGAGGCCGGGGTG-3′ (SEQ IDNO. 28) and 5′-CTTCTGCAAGTCCCAGAGCCAGTGTGC-3′ (SEQ ID NO. 24)) were usedto amplify a 1736 nt portion of the CD39-L2 mRNA sequence. Primersspecific to human β-actin gene (5′CGGGATCCCTGTGCTACGTCGCCCTGGAC-3′ (SEQID NO. 38) and 5′-CGGAATTCACTGGCGCAGGCGGTGATCTCCTT-3′ (SEQ ID NO. 39))were used to amplify a 315 nt fragment of the gene to serve as positivecontrol for the cDNA libraries. The PCR conditions were as follows: 96°C. for 2.5 minutes (I cycle); 96° C. for 45 seconds, 60° C. for 45seconds, and 72° C. for 2.5 minutes (3 cycles); 94° C. for 30 seconds,60° C. for 30 seconds and 72° C. for 2.5 minutes (30 cycles); and 72° C.for 6 minutes (1 cycle). Amplified products were separated on a 1.2%agarose gel. Out of 15 libraries tested, only adult heart and fetalbrain yielded bands. The bands were sequenced to confirm primerspecificity. The heart derived product encodes the functional CD39-L2sequence that has been demonstrated to possess ADPase activity. Thefetal brain-derived product represents a variant with 43 bp ofadditional sequence added to exon 14, resulting in a frame shift of thelast 260 bp of coding sequence (SEQ ID NO: 45).

[0341] Other splice variants have also been identified in adult brain,adult bladder, adult rectum and adult mammary gland (SEQ ID NO: 46),adult brain, umbilical cord, adult thymus and adult mammary gland (SEQID NO: 47), adult brain (SEQ ID NO: 48), adult brain (SEQ ID NO: 49),adult brain, adult bladder, adult thymus, adult mammary gland, adultkidney, lung tumor, adult adrenal gland and adult thalamus (SEQ ID NO:50), adult adrenal gland (SEQ ID NO: 51), adult colon and adult thymus(SEQ ID NO: 52), fetal lung (SEQ ID NO: 53), and adult thymus (SEQ IDNO: 54).

[0342] For the Northern blot analysis, a human Multiple Tissue Northern(MTN™) blot was purchased from CLONTECH. A 1008 nt portion of CD39-L2gene, corresponding to the 3′UTR, was amplified by PCR using genespecific primers (5′-CATCCTGAGGAGCCACAGCAC-3′ (SEQ ID NO: 40);5′-AGGTTCAGCTCGTGCCGGGCA-3′ (SEQ ID NO: 41)). A 315 nt portion of humanβ-actin gene was amplified with the same gene-specific primers asdescribed above. The probes used in the hybridization were generated bylabeling the PCR products with the Prime-It@II Random Primer LabelingKit from Stratagene (La Jolla, Calif.) in the presence of [α-³³P]dCTP.The hybridization was performed using the ExpressHyb™hybridizationsolution from CLONTECH according to the instructions of themanufacturer. The heart displayed the highest level of expression amongall the tissues sampled, being at least 2-fold higher than that ofbrain. The other tissues, including placenta, lung, liver, skeletalmuscle, kidney and pancreas, showed a negligible amount of expression.The high levels of expression of CD39-L2 in heart implicates thisprotein as a regulator of platelet aggregation in this organ.

EXAMPLE 16 Cellular Localization and Characterization of CD39-L2

[0343] A. Cellular Localization

[0344] Western blot analysis was performed on COS cells transfected withthe plasmid described in Example 15 above to determine the cellularlocalization of CD39-L2. To detect myc epitope tagged recombinantproteins, the blot was incubated with a 2000-fold dilution of theanti-c-myc monoclonal antibody (Invitrogen) at room temperature for twohours. The secondary antibody (anti-mouse Ig AP conjugate) was diluted1000-fold and incubated for 1-2 hours at room temperature. Boundantibody was detected by using Sigma Fast™ 5-bromo-4-chloro-3indolylphosphate/nitro blue tetrazolium (BCIP/NTP) as the alkaline phosphatasesubstrate according to instructions of the manufacturer.

[0345] The recombinant protein was detected in the media and themembrane fractions of the CD39-L2 transfected cells, but not in thecytosolic fraction or control transfections. The relative bandintensities suggest that the majority of the recombinant CD39-L2 proteinis secreted into the media and a fraction resides in the membrane. Thepredicted molecular weight of unprocessed CD39-L2 is 53 kD. However,both the membrane and secreted fractions displayed slower mobility bySDS/PAGE than that predicted by its amino acid content, suggestingpost-translational modification.

[0346] B. Secretion of CD39-L2

[0347] To confirm that recombinant CD39-L2 is secreted, the cellularlocalization was performed using increasing amounts of brefeldin A, aninhibitor of translocation of secretory proteins from the endoplasmicreticulum to the Golgi apparatus. Briefly, brefeldin A was dissolved inethanol and added to the transfected cells 48 hours after transfection.Both control and brefeldin A-treated cells were washed once with PBS andincubated for 8 hours in serum-free DMEM with none or varying dosages ofbrefeldin A. For the treated cells, recombinant CD39-L2 in the mediadecreased in a brefeldin A dose dependent manner. Correspondingly,recombinant CD39-L2 also accumulated in the cytosol in a dose dependentmanner. Therefore, it can be inferred that recombinant CD39-L2 secretionfollows the conventional cellular secretory pathway.

[0348] Flow cytometric analysis was used to determine if recombinantCD39-L2 is expressed on cell surfaces. COS-7 cells were transfected asdescribed above with either pcDNA3.1/myc-His(+)A or pCD39-L2myc-HIS.After 72 hours of transfection, the cells were washed twice with PBS,and dislodged with 10 ml EDTA in PBS. Cells were pelleted bycentrifugation at 300 g for five minutes, washed with PBS andresuspended in binding buffer (PBS containing 3% FBS and 0.02% sodiumazide) at a concentration of 1×10⁶ cells per 100 μl. The cells werefirst stained with 20 μg/ml of monoclonal anti-myc antibody for 30minutes at 4° C. The cells were then washed with binding buffer andstained with 20 μg/ml of R-phycoerythrin conjugated goat anti-mouse IgGantibody (Molecular Probes, Eugene, Oreg.). After washing with bindingbuffer, the cells were resuspended in 1 ml of binding buffer andanalyzed on the FACScalibur flow cytometer (Becton DickinsonImmunocytometry Systems, San Jose, Calif.).

[0349] Expression of cell surface recombinant CD39-L2 was found only oncells transfected with pCD39-L2myc-His, while cells from the controltransfection showed no antibody binding.

[0350] These results of sections A and B demonstrate that recombinantCD39L2 is present in the membrane fraction and is expressed on the cellsurface as shown by flow cytometry. These data suggest that CD39L2 is acell membrane-bound protein. However, the results also show thatrecombinant CD39L2 is secreted and soluble. One possible explanation isthat the soluble form is derived from the transmembrane form viaproteolytic event, as in the cases of tumor necrosis factor and othermembrane-derived soluble proteins. Thus, the active soluble form ofCD39L2 may be missing at least about 60 amino acids from the N-terminusof the amino acid sequence set forth in SEQ ID NO: 27.

[0351] C. N-Linked Glycosylation of CD39-L2

[0352] To determine whether CD39-L2 is N-linked glycosylated, CD39-L2obtained from COS cell conditioned media was tested with Peptide:N-glycosidase F (New England Biolabs, Beverly, Mass.) according tomanufacturer's instructions. Briefly, CD39-L2 was denatured withdenaturing buffer (0.5% SDS, 1% β-mercaptoethanol) at 100° C. for tenminutes. The CD39-L2 was then incubated with Peptide: N-glycosidase F inG7 buffer (50 mM sodium phosphate, pH 7.5 @25° C.) supplemented with 1%NP-40 at 37° C. for two hours. Another sample of CD39-L2 was treatedidentically but without the Peptide: N-glycosidase F. Both samples weresubjected to SDS-PAGE and were detected by immunodetection usingantibodies against the myc-6His epitope.

[0353] The sample treated with glycosidase exhibited faster migration onSDS-PAGE than the untreated sample, indicating a reduction in molecularweight. Therefore, CD39-L2 contains N-linked glycosylation that can beenzymatically removed by a glycosidase. There are two potentialN-glycosylation sites (Asn-X-Ser/Thr) in the predicted protein sequence.

EXAMPLE 17 Characterization of CD39-L2 Activity

[0354] A. Assay of Nucleotidase Activity

[0355] Recombinant CD39L2 expressed from transiently transfected COScells was tested for its NDPase and NTPase activities. CD39-L2 proteinwas assayed for ADPase activity in the presence of different kinds ofinhibitors of ADPases. Control ecto-apyrase activity was determined withprotein tethered to the Nickel-NTA resin. Both assays were performed asdescribed in Example 12 above except the protein was in buffer Acontaining 1 mM EGTA and 3 mM CaCl₂. The assay was started by adding ADPto 1 mM followed by a 30 minute incubation at 37° C.

[0356] As shown by the results in Table 3 below, CD39-L2 is notinhibited by inhibitors of vacuolar adenosine triphospatase (ATPases)(NEM), mitochondrial ATPase (N₃ ⁻) and Na⁻, K⁻ ATPase (oubain). Aninhibitor of adenylate kinase (Ap5A) did not inhibit activity, while aninhibitor of phosphatases (F⁻) partially inhibited activity. Metalchelators (EDTA and EGTA) inhibited CD39-L2 activity therebydemonstrating that CD39-L2 activity is dependent on divalent cations.Assays repeated at higher levels of substrate (e.g. 15 mM ADP with 15 mMCaCl₂) produced similar results. In addition, CD39L2 ADPase activity at15 mM ADP was stimulated upon addition of divalent cations; addition ofMg² (MgCl₂ added to 15 mM) stimulates activity 8-fold and Ca²⁻ (CaCl₂added to 15 mM) stimulates activity 17-fold.

[0357] These results indicate that CD39L2 exhibits characteristicfeatures of ecto-apyrases. TABLE 3 Inhibition of CD39-L2 activityINHIBITORS % OF CONTROL Control 100 ± 3  Ouabain (1 mM) 101 ± 9  NEM (10mM) 88.4 ± 13   N₃ (1 mM) 90 ± 13 F (10 mM) 63 ± 9  Ap5A (10 μM) 87 ± 11EGTA (2 mM) 34 ± 10 EDTA (2 mM) 18.4 ± 9  

[0358] The nucleotide specificity of CD39-L2 was also assayed asdescribed in Example 12. The CD39-L2 activity was determined withprotein tethered to the Ni-NTA resin. The protein was in assay buffer Acontaining 1 mM EGTA, 3 mM CaCl₂ and 3 mM MgCl₂. The assay was startedby adding the nucleotides to a final concentration of 1 mM. The sampleswere assayed at 37° C. for 30 minutes. Results are shown in Table 4below, wherein values are expressed relative to ADP. TABLE 4 SubstrateSpecificity of CD39-L2 NUCLEOTIDE % OF CONTROL ADP 100 ± 8  ATP 16 ± 2AMP 0.6 ± 1  CTP 44 ± 4 GTP 39 ± 1 UTP 13 ± 1 CDP 282 ± 18 GDP 338 ± 52UDP 303 ± 5 

[0359] CD39L2 shows a substrate specificity for ADP over ATP and theother triphosphate nucleotides. Assays repeated with 15 mM ADP and 15 mMCaCl2 confirmed the substrate specificity of CD39L2 for NDP over NTPs;enzymatic activities with CDP, GDP and UDP as substrates were observed,respectively, to be 73%, 280% and 228% relative to ADP hydrolysis. Theseresults confirm that CD39-L2 along with CD39-L4 define a new class ofE-type apyrase in humans with a specificity for NDPs as enzymaticsubstrates.

[0360] The effect of excess free Ca²1 ions, which inhibits the activityof CD39 and other ecto-apyrases, on ADPase activity of CD39L2 was alsodetermined. A titration analysis of Ca²⁺ concentration on ADPaseactivity was performed at various ADP concentrations. The protein was inassay buffer A containing concentrations of CaCl₂ varying from 2 mM to15 mM. The assay was started by adding the required amount of ADP atconcentrations varying from 2 mM to 15 mM. Each sample was assayed intriplicate at 37 C for 20 minutes and values were expressed relative tothe reactions containing 15 mM CaCl₂ and 15 mM ADP. Results showed thathigh Ca²⁺ concentration had no inhibitory effect on CD39L2 ADPaseactivity at low substrate concentration. For high substrateconcentrations, a correspondingly high Ca²⁻ concentration was requiredfor full activation.

[0361] B. Determination of Kinetic Characteristics of CD39-1,2 ADPHydrolysis

[0362] To determine the kinetic characteristics of ADP hydrolysis byCD39L2, recombinant soluble CD39L2 obtained by low pH elution ofCD39L2-bound Ni-NTA resin was prepared. Recombinant CD39L2 was elutedoff the Ni-NTA resin with acidic elution buffer (0.1 M sodium acetate.pH 4.5, 0.3 M NaCl and 0.2 mg/ml inactivated peroxidase (Sigma) ascarrier protein). Tris HCl, pH 9, was then added to a finalconcentration of 70 mM to neutralize the eluted protein sample. Proteinswere concentrated 2-fold while small solute concentrations were reducedby 80% using Microcon concentrators. Reactions were carried out in thepresence of the apyrase assay buffer with 15 mM CaCl₂, 1 mM ouabain, 10mM NEM, 10 μM Ap5A and concentrations of ADP varying from 0.75 mM to 18mM, and incubated for 15 min at 37° C. The amount of phosphate releasedfrom the reaction was assayed as described above. The amount of theeluted protein used in the reaction was determined by visualizing withGelcode® Blue (Pierce) staining following SDS-PAGE, and comparing to aseries of bovine serum albumin standards of known concentrations.Because the CD39L2 protein is not purified to homogenicity, the locationof the CD39-L2 band was identified by immunodetection on a duplicatedlane from the same gel.

[0363] Assays were carried out as described above. The rate of productrelease was found to be linear within the first 20 minutes of reaction,so therefore the initial velocity V₀ is taken to be the rate of reactionover the first 15 minutes. V₀ was determined over a range of substrateconcentrations, and was found to respond in a sigmoidal fashion,indicating positive cooperativity. Each data point represented anaverage of three separate experiments. Curve-fitting of Hill's Equation{V_(o)=(V_(max)[S]^(n))/(K+[S]^(n))} to the data points was performed byusing the DeltaGraph® 4.0 software (SPSS Inc., Chicago, Ill.) and theresulting R² value was 0.995. The cooperativity, n, was determined to be2.48, V_(max)=1028 pmol/min and K=364.5. The substrate at half-maximalvelocity [S]_(0.5)=K^(t/n)=10.6 mM. The amount of partially purifiedCD39L2 protein used in each reaction was estimated to be 3.5 ng so thatthe specific activity of V_(max) was calculated to be at least 290μmol/min/mg.

[0364] To date, CD39L2 is the only ecto-apyrase that demonstratespositive cooperativity. In contrast, CD39L4 ADPase activity does notappear to possess cooperativity. The positive cooperativity for ADPaseactivity displayed by the CD39L2 protein indicates that this protein isforming multimers and is predicted to be most active as a tetramer.

[0365] Optimal ADP concentrations for CD39L2 are estimated to be in themillimolar range. However, levels of ADP in the circulation have beenestimated to be in the low micromolar range. This suggests that CD39 L2would be effective for preventing or reducing a sudden rise in ADPlevels e.g., due to release of ADP from dense granules inside platelets(which have been shown to contain high concentrations of ADP estimatedto be up to 0.5 M) within the heart or coronary circulation duringinjury or trauma. Relatively high concentrations of ADP in themicroenvironment of an injury could be closer to the levels where CD39L2functions optimally. The estimated specific activity of 290 μmol/min/mgfor CD39L2 is significantly higher than that reported for the truncatedsoluble CD39, which is 11 μmol/min/mg at saturation [Gayle et al., J.Clin. Invest., 101, 1851-59 (1998)]. Taken together, the positivecooperativity and high specific activity of CD39L2 suggests that its canfunction to hydrolyze large amounts of ADP rapidly. These features areexpected to be useful in reducing the incidence and/or recurrence ofvascular occlusions caused by excessive platelet aggregation,particularly during traumas or insults.

EXAMPLE 18 Determination of CD39-L4 and CD39-L2 Expression Using In SituHybridization and Immunohistochemistry

[0366] A. In situ Hybridization and Immunohistochemistry of CD39-L4 inKidney

[0367] Tissues were hybridized with DIG-labeled riboprobes derived fromCD39L4 coding sequence nucleotides. A 298 nt fragment of the CD39L4 cDNA3′-untranslated region was amplified by PCR with oligonucleotide primers246D13 and 246D4 (5′-ATCCTGGACTTGAGCCTAGAG-3′, SEQ ID NO: 34 and5′-CTGATATTGATGGGTCTTGGG-3′, SEQ ID NO: 35). The fragment was subclonedinto the pCR™ II-TOPO plasmid (Invitrogen) and sense and antisense RNAwere synthesized. The probe was labeled using the digoxigenin labelingkit supplied by Boehringer-Mannheim as described in the manufacturersprotocol. Automated in situ hybridization was performed by QualTekMolecular Labs (Santa Barbara, Calif.) using a modified version of apreviously published procedure (Myers, J. A., et al., (1995) J. Surg.Path. 1, 191-203). The Ventana Medical Systems, Inc. (Tucson, Ariz.)TechMate™ Automated Staining System was used for this procedure. Alltissues were fixed in 10% neutral buffered formalin, paraffin-embeddedand cut into 4 μm thick sections. Sections were placed onto Ventana'sChemMate™ Capillary Gap Slides (POP075).

[0368] Staining of kidney sections revealed that specific cell typeshybridized with the antisense probe but not the sense probe in a highlyspecific manner. The staining of the glomerulus revealed that theepithelia of the Bowman's capsule, podocyte epithelia and mesangialcells were specifically stained. The expression of the CD39L4 protein inthis region could be necessary to prevent platelet aggregation in theBowman's capsule because platelets become highly concentrated in thisparticular region as water and ions are filtered from the blood. Thebloody region within the kidney showed staining of white blood cells,presumably macrophages. This staining is consistent with previousstudies where a macrophage cDNA library showed expression of the CD39L4cDNA. CD39L4 staining was also found in some tubule epithelial cells inthe kidney.

[0369] Immunohistochemistry on the same tissue was conducted usingpolyclonal anti-CD39L4 antibody prepared by immunizing rabbits withpeptide 246A (EVAKDSIPRSHWKK, SEQ ID NO: 43, corresponding to aminoacids 109 to 122 of SEQ ID NO: 3) conjugated to keyhole limpethemacyanin (KLH), using conventional methods [see, e.g., Harlow et al.,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y. (1998)]. The antibodies were affinity purified usingpeptide 246A sequence with an extra Cys residue at the C-terminus toallow coupling to a Sulfo link coupling gel (Pierce).

[0370] Results showed that the same cell types that expressed CD39L4mRNA also expressed CD39L4 protein.

[0371] B. In situ Hybridization and Immunohistochemistry of CD39-L2 inHeart

[0372] Tissues were hybridized with DIG-labeled riboprobes derived fromCD39L2 coding sequence nucleotides 944-1134. Riboprobes were preparedusing the DIG RNA Labeling Kit (Roche Molecular Biochemicals) asdescribed by the instructions of the manufacturer. The 186 nt fragmentof the CD39L2 cDNA was amplified by PCR with oligonucleotide primersL2RNA3 and L2RNA2 (5′-GGATGGAAAGGAGTTGGTCAG-3′, SEQ ID NO: 36 and5′-GTCCACATGCTTCACTTCCTC-3′ SEQ ID NO: 37). The fragment was subclonedinto the pCR™ II-TOPO plasmid (Invitrogen) and sense and antisense RNAwere synthesized and labeled as described above. Automated in situhybridization was performed as described above.

[0373] Staining of heart sections revealed that specific cell typeshybridized with the antisense probe but not the sense probe in a highlyspecific manner. The cardiac muscle cells as well as capillaryendothelial cells and white blood cells within a blood vessel showedspecific staining. This staining is consistent with previous studieswhere a heart cDNA library showed robust expression of the CD39L2 cDNA.

[0374] Immunohistochemistry on the same tissues is conducted usinganti-CD39L2 antibody prepared by immunizing rabbits with peptide 102B(TRPPRETPTLTHET, SEQ ID NO: 44, corresponding to amino acids 121 to 134in SEQ ID NO: 27) conjugated to KLH, using conventional methods [see,e.g., Harlow et al., “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y. (1998)]. Results areexpected to confirm that the same cell types that express mRNA alsoexpressed CD39L2 protein.

[0375] This in situ hybridization data is consistent with aphysiological role for CD39-L4 and CD39-L2 in regulating plateletaggregation and hemostasis. Further in situ hybridization may be carriedout to confirm this activity.

[0376] The present invention is not to be limited in scope by theexemplified embodiments which are intended as illustrations of singleaspects of the invention, and compositions and methods which arefunctionally equivalent are within the scope of the invention. Indeed,numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe present preferred embodiments. Consequently, the only limitationswhich should be placed upon the scope of the invention are those whichappear in the appended claims. All references cited within the body ofthe instant specification are hereby incorporated by reference in theirentirety.

1 54 1 300 DNA Homo sapiens 1 ggcatattag cttgggttac tgtgaattttctgacaggtc agctgcatgg ccacagacag 60 gagactgtgg ggaccttgga cctagggggagcctccaccc aaatcacgtt cctgccccag 120 tttgagaaaa ctctggaaca aactcctaggggctacctca cttcctttga gatgtttaac 180 agcacttata agctctatac acatagttacctgggatttg gattgaaagc tgcaagacta 240 gcaaccctgg gagccctgga gacagaagggactgatgggc acactttccg gagtgcctgt 300 2 1799 DNA Homo sapiens CDS(246)..(1529) misc_feature (1718) n = adenine or guanine or cytosine orthymidine 2 gcgggctgcc gcgcaagggt ggcgcgcgcg cgttttcctt gttcctggtcaacaaagaaa 60 tgtggagtgt cttggctgaa tcctcataca gacaagatca ttatggtgctgttaggttga 120 aaaagtgata taataaagga accaaggaga aaattcagaa ggaaagaaaaaattgcctct 180 gcaggtgtgc gagcaggatt gcttctgcaa caaaagcctc cacccagccacatcttggga 240 aaaga atg gcc act tct tgg ggc aca gtc ttt ttc atg ctg gtggta tcc 290 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser1 5 10 15 tgt gtt tgc agc gct gtc tcc cac agg aac cag cag act tgg tttgag 338 Cys Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu20 25 30 ggt atc ttc ctg tct tcc atg tgc ccc atc aat gtc agc gcc agc acc386 Gly Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr 3540 45 ttg tat gga att atg ttt gat gca ggg agc act gga act cga att cat434 Leu Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His 5055 60 gtt tac acc ttt gtg cag aaa atg cca gga cag ctt cca att cta gaa482 Val Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu 6570 75 ggg gaa gtt ttt gat tct gtg aag cca gga ctt tct gct ttt gta gat530 Gly Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp 8085 90 95 caa cct aag cag ggt gct gag acc gtt caa ggg ctc tta gag gtg gcc578 Gln Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala 100105 110 aaa gac tca atc ccc cga agt cac tgg aaa aag acc cca gtg gtc cta626 Lys Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu 115120 125 aag gca aca gca gga cta cgc tta ctg cca gaa cac aaa gcc aag gct674 Lys Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys Ala 130135 140 ctg ctc ttt gag gta aag gag atc ttc agg aag tca cct ttc ctg gta722 Leu Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe Leu Val 145150 155 cca aag ggc agt gtt agc atc atg gat gga tcc gac gaa ggc ata tta770 Pro Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp Glu Gly Ile Leu 160165 170 175 gct tgg gtt act gtg aat ttt ctg aca ggt cag ctg cat ggc cacaga 818 Ala Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His Gly His Arg180 185 190 cag gag act gtg ggg acc ttg gac cta ggg gga gcc tcc acc caaatc 866 Gln Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile195 200 205 acg ttc ctg ccc cag ttt gag aaa act ctg gaa caa act cct aggggc 914 Thr Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly210 215 220 tac ctc act tcc ttt gag atg ttt aac agc act tat aag ctc tataca 962 Tyr Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr225 230 235 cat agt tac ctg gga ttt gga ttg aaa gct gca aga cta gca accctg 1010 His Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu240 245 250 255 gga gcc ctg gag aca gaa ggg act gat ggg cac act ttc cggagt gcc 1058 Gly Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr Phe Arg SerAla 260 265 270 tgt tta ccg aga tgg ttg gaa gca gag tgg atc ttt ggg ggtgtg aaa 1106 Cys Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly ValLys 275 280 285 tac cag tat ggt ggc aac caa gaa ggg gag gtg ggc ttt gagccc tgc 1154 Tyr Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly Phe Glu ProCys 290 295 300 tat gcc gaa gtg ctg agg gtg gta cga gga aaa ctt cac cagcca gag 1202 Tyr Ala Glu Val Leu Arg Val Val Arg Gly Lys Leu His Gln ProGlu 305 310 315 gag gtc cag aga ggt tcc ttc tat gct ttc tct tac tat tatgac cga 1250 Glu Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr AspArg 320 325 330 335 gct gtt gac aca gac atg att gat tat gaa aag ggg ggtatt tta aaa 1298 Ala Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly IleLeu Lys 340 345 350 gtt gaa gat ttt gaa aga aaa gcc agg gaa gtg tgt gataac ttg gaa 1346 Val Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp AsnLeu Glu 355 360 365 aac ttc acc tca ggc agt cct ttc ctg tgc atg gat ctcagc tac atc 1394 Asn Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu SerTyr Ile 370 375 380 aca gcc ctg tta aag gat ggc ttt ggc ttt gca gac agcaca gtc tta 1442 Thr Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser ThrVal Leu 385 390 395 cag ctc aca aag aaa gtg aac aac ata gag acg ggc tgggcc ttg ggg 1490 Gln Leu Thr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp AlaLeu Gly 400 405 410 415 gcc acc ttt cac ctg ttg cag tct ctg ggc atc tcccat tgaggccacg 1539 Ala Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His420 425 tacttccttg gagacctgca tttgccaaca cctttttaag gggaggagagagcacttagt 1599 ttctgaacta gtctggggac atcctggact tgagcctaga gattwrgttaattaascggc 1659 cgagcttatc cttwatragg taatttactt gcmtggccgc gtttacacgtcgtgatggna 1719 aacctgcgtc ccaactaacg cttgasamat ccccttcgca gctgcgataccaaaagccga 1779 cgacgccttc cacagtgcca 1799 3 428 PRT Homo sapiens 3 MetAla Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 7580 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 9095 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu Lys115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys AlaLeu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe LeuVal Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp GluGly Ile Leu Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly Gln LeuHis Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu Gly GlyAla Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys Thr LeuGlu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met Phe AsnSer Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly Phe GlyLeu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu Thr GluGly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro Arg TrpLeu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln Tyr GlyGly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 Ala GluVal Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr IleThr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr ValLeu Gln 385 390 395 400 Leu Thr Lys Lys Val Asn Asn Ile Glu Thr Gly TrpAla Leu Gly Ala 405 410 415 Thr Phe His Leu Leu Gln Ser Leu Gly Ile SerHis 420 425 4 1287 DNA Homo sapiens CDS (1)..(1284) 4 atg gcc act tcttgg ggc aca gtc ttt ttc atg ctg gtg gta tcc tgt 48 Met Ala Thr Ser TrpGly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15 gtt tgc agc gctgtc tcc cac agg aac cag cag act tgg ttt gag ggt 96 Val Cys Ser Ala ValSer His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30 atc ttc ctg tct tccatg tgc ccc atc aat gtc agc gcc agc acc ttg 144 Ile Phe Leu Ser Ser MetCys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45 tat gga att atg ttt gatgca ggg agc act gga act cga att cat gtt 192 Tyr Gly Ile Met Phe Asp AlaGly Ser Thr Gly Thr Arg Ile His Val 50 55 60 tac acc ttt gtg cag aaa atgcca gga cag ctt cca att cta gaa ggg 240 Tyr Thr Phe Val Gln Lys Met ProGly Gln Leu Pro Ile Leu Glu Gly 65 70 75 80 gaa gtt ttt gat tct gtg aagcca gga ctt tct gct ttt gta gat caa 288 Glu Val Phe Asp Ser Val Lys ProGly Leu Ser Ala Phe Val Asp Gln 85 90 95 cct aag cag ggt gct gag acc gttcaa ggg ctc tta gag gtg gcc aaa 336 Pro Lys Gln Gly Ala Glu Thr Val GlnGly Leu Leu Glu Val Ala Lys 100 105 110 gac tca atc ccc cga agt cac tggaaa aag acc cca gtg gtc cta aag 384 Asp Ser Ile Pro Arg Ser His Trp LysLys Thr Pro Val Val Leu Lys 115 120 125 gca aca gca gga cta cgc tta ctgcca gaa cac aaa gcc aag gct ctg 432 Ala Thr Ala Gly Leu Arg Leu Leu ProGlu His Lys Ala Lys Ala Leu 130 135 140 ctc ttt gag gta aag gag atc ttcagg aag tca cct ttc ctg gta cca 480 Leu Phe Glu Val Lys Glu Ile Phe ArgLys Ser Pro Phe Leu Val Pro 145 150 155 160 aag ggc agt gtt agc atc atggat gga tcc gac gaa ggc ata tta gct 528 Lys Gly Ser Val Ser Ile Met AspGly Ser Asp Glu Gly Ile Leu Ala 165 170 175 tgg gtt act gtg aat ttt ctgaca ggt cag ctg cat ggc cac aga cag 576 Trp Val Thr Val Asn Phe Leu ThrGly Gln Leu His Gly His Arg Gln 180 185 190 gag act gtg ggg acc ttg gaccta ggg gga gcc tcc acc caa atc acg 624 Glu Thr Val Gly Thr Leu Asp LeuGly Gly Ala Ser Thr Gln Ile Thr 195 200 205 ttc ctg ccc cag ttt gag aaaact ctg gaa caa act cct agg ggc tac 672 Phe Leu Pro Gln Phe Glu Lys ThrLeu Glu Gln Thr Pro Arg Gly Tyr 210 215 220 ctc act tcc ttt gag atg tttaac agc act tat aag ctc tat aca cat 720 Leu Thr Ser Phe Glu Met Phe AsnSer Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 agt tac ctg gga ttt ggattg aaa gct gca aga cta gca acc ctg gga 768 Ser Tyr Leu Gly Phe Gly LeuLys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 gcc ctg gag aca gaa gggact gat ggg cac act ttc cgg agt gcc tgt 816 Ala Leu Glu Thr Glu Gly ThrAsp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 tta ccg aga tgg ttg gaagca gag tgg atc ttt ggg ggt gtg aaa tac 864 Leu Pro Arg Trp Leu Glu AlaGlu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 cag tat ggt ggc aac caagaa ggg gag gtg ggc ttt gag ccc tgc tat 912 Gln Tyr Gly Gly Asn Gln GluGly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 gcc gaa gtg ctg agg gtggta cga gga aaa ctt cac cag cca gag gag 960 Ala Glu Val Leu Arg Val ValArg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320 gtc cag aga ggt tccttc tat gct ttc tct tac tat tat gac cga gct 1008 Val Gln Arg Gly Ser PheTyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330 335 gtt gac aca gac atgatt gat tat gaa aag ggg ggt att tta aaa gtt 1056 Val Asp Thr Asp Met IleAsp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340 345 350 gaa gat ttt gaa agaaaa gcc agg gaa gtg tgt gat aac ttg gaa aac 1104 Glu Asp Phe Glu Arg LysAla Arg Glu Val Cys Asp Asn Leu Glu Asn 355 360 365 ttc acc tca ggc agtcct ttc ctg tgc atg gat ctc agc tac atc aca 1152 Phe Thr Ser Gly Ser ProPhe Leu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 gcc ctg tta aag gatggc ttt ggc ttt gca gac agc aca gtc tta cag 1200 Ala Leu Leu Lys Asp GlyPhe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395 400 ctc aca aag aaagtg aac aac ata gag acg ggc tgg gcc ttg ggg gcc 1248 Leu Thr Lys Lys ValAsn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415 acc ttt cac ctgttg cag tct ctg ggc atc tcc cat tga 1287 Thr Phe His Leu Leu Gln Ser LeuGly Ile Ser His 420 425 5 428 PRT Homo sapiens 5 Met Ala Thr Ser Trp GlyThr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15 Val Cys Ser Ala ValSer His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30 Ile Phe Leu Ser SerMet Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45 Tyr Gly Ile Met PheAsp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60 Tyr Thr Phe Val GlnLys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 75 80 Glu Val Phe AspSer Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 90 95 Pro Lys Gln GlyAla Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100 105 110 Asp Ser IlePro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu Lys 115 120 125 Ala ThrAla Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys Ala Leu 130 135 140 LeuPhe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe Leu Val Pro 145 150 155160 Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp Glu Gly Ile Leu Ala 165170 175 Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His Gly His Arg Gln180 185 190 Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln IleThr 195 200 205 Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro ArgGly Tyr 210 215 220 Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys LeuTyr Thr His 225 230 235 240 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala ArgLeu Ala Thr Leu Gly 245 250 255 Ala Leu Glu Thr Glu Gly Thr Asp Gly HisThr Phe Arg Ser Ala Cys 260 265 270 Leu Pro Arg Trp Leu Glu Ala Glu TrpIle Phe Gly Gly Val Lys Tyr 275 280 285 Gln Tyr Gly Gly Asn Gln Glu GlyGlu Val Gly Phe Glu Pro Cys Tyr 290 295 300 Ala Glu Val Leu Arg Val ValArg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320 Val Gln Arg Gly SerPhe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330 335 Val Asp Thr AspMet Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340 345 350 Glu Asp PheGlu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn 355 360 365 Phe ThrSer Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 AlaLeu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395400 Leu Thr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405410 415 Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His 420 425 6 1287DNA Homo sapiens CDS (1)..(1284) 6 atg gcc act tct tgg ggc aca gtc tttttc atg ctg gtg gta tcc tgt 48 Met Ala Thr Ser Trp Gly Thr Val Phe PheMet Leu Val Val Ser Cys 1 5 10 15 gtt tgc agc gct gtc tcc cac agg aaccag cag act tgg ttt gag ggt 96 Val Cys Ser Ala Val Ser His Arg Asn GlnGln Thr Trp Phe Glu Gly 20 25 30 atc ttc ctg tct tcc atg tgc ccc atc aatgtc agc gcc agc acc ttg 144 Ile Phe Leu Ser Ser Met Cys Pro Ile Asn ValSer Ala Ser Thr Leu 35 40 45 tat gga att atg ttt gat gca ggg agc act ggaact cga att cat gtt 192 Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly ThrArg Ile His Val 50 55 60 tac acc ttt gtg cag aaa atg cca gga cag ctt ccaatt cta gaa ggg 240 Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro IleLeu Glu Gly 65 70 75 80 gaa gtt ttt gat tct gtg aag cca gga ctt tct gctttt gta gat caa 288 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala PheVal Asp Gln 85 90 95 cct aag cag ggt gct gag acc gtt caa ggg ctc tta gaggtg gcc aaa 336 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu ValAla Lys 100 105 110 gac tca atc ccc cga agt cac tgg aaa aag acc cca gtggtc cta aag 384 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val ValLeu Lys 115 120 125 gca aca gca gga cta cgc tta ctg cca gaa cac aaa gccaag gct ctg 432 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala LysAla Leu 130 135 140 ctc ttt gag gta aag gag atc ttc agg aag tca cct ttcctg gta cca 480 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe LeuVal Pro 145 150 155 160 aag ggc agt gtt agc atc atg act gga caa gac gaaggc ata ttc gct 528 Lys Gly Ser Val Ser Ile Met Thr Gly Gln Asp Glu GlyIle Phe Ala 165 170 175 tgg gtt act gtg aat ttt ctg aca ggt cag ctg catggc cac aga cag 576 Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His GlyHis Arg Gln 180 185 190 gag act gtg ggg acc ttg gac cta ggg gga gcc tccacc caa atc acg 624 Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser ThrGln Ile Thr 195 200 205 ttc ctg ccc cag ttt gag aaa act ctg gaa caa actcct agg ggc tac 672 Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr ProArg Gly Tyr 210 215 220 ctc act tcc ttt gag atg ttt aac agc act tat aagctc tat aca cat 720 Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys LeuTyr Thr His 225 230 235 240 agt tac ctg gga ttt gga ttg aaa gct gca agacta gca acc ctg gga 768 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg LeuAla Thr Leu Gly 245 250 255 gcc ctg gag aca gaa ggg act gat ggg cac actttc cgg agt gcc tgt 816 Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr PheArg Ser Ala Cys 260 265 270 tta ccg aga tgg ttg gaa gca gag tgg atc tttggg ggt gtg aaa tac 864 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe GlyGly Val Lys Tyr 275 280 285 cag tat ggt ggc aac caa gaa ggg gag gtg ggcttt gag ccc tgc tat 912 Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly PheGlu Pro Cys Tyr 290 295 300 gcc gaa gtg ctg agg gtg gta cga gga aaa cttcac cag cca gag gag 960 Ala Glu Val Leu Arg Val Val Arg Gly Lys Leu HisGln Pro Glu Glu 305 310 315 320 gtc cag aga ggt tcc ttc tat gct ttc tcttac tat tat gac cga gct 1008 Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser TyrTyr Tyr Asp Arg Ala 325 330 335 gtt gac aca gac atg att gat tat gaa aagggg ggt att tta aaa gtt 1056 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys GlyGly Ile Leu Lys Val 340 345 350 gaa gat ttt gaa aga aaa gcc agg gaa gtgtgt gat aac ttg gaa aac 1104 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val CysAsp Asn Leu Glu Asn 355 360 365 ttc acc tca ggc agt cct ttc ctg tgc atggat ctc agc tac atc aca 1152 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met AspLeu Ser Tyr Ile Thr 370 375 380 gcc ctg tta aag gat ggc ttt ggc ttt gcagac agc aca gtc tta cag 1200 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala AspSer Thr Val Leu Gln 385 390 395 400 ctc aca aag aaa gtg aac aac ata gagacg ggc tgg gcc ttg ggg gcc 1248 Leu Thr Lys Lys Val Asn Asn Ile Glu ThrGly Trp Ala Leu Gly Ala 405 410 415 acc ttt cac ctg ttg cag tct ctg ggcatc tcc cat tga 1287 Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His 420425 7 428 PRT Homo sapiens 7 Met Ala Thr Ser Trp Gly Thr Val Phe Phe MetLeu Val Val Ser Cys 1 5 10 15 Val Cys Ser Ala Val Ser His Arg Asn GlnGln Thr Trp Phe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Met Cys Pro Ile AsnVal Ser Ala Ser Thr Leu 35 40 45 Tyr Gly Ile Met Phe Asp Ala Gly Ser ThrGly Thr Arg Ile His Val 50 55 60 Tyr Thr Phe Val Gln Lys Met Pro Gly GlnLeu Pro Ile Leu Glu Gly 65 70 75 80 Glu Val Phe Asp Ser Val Lys Pro GlyLeu Ser Ala Phe Val Asp Gln 85 90 95 Pro Lys Gln Gly Ala Glu Thr Val GlnGly Leu Leu Glu Val Ala Lys 100 105 110 Asp Ser Ile Pro Arg Ser His TrpLys Lys Thr Pro Val Val Leu Lys 115 120 125 Ala Thr Ala Gly Leu Arg LeuLeu Pro Glu His Lys Ala Lys Ala Leu 130 135 140 Leu Phe Glu Val Lys GluIle Phe Arg Lys Ser Pro Phe Leu Val Pro 145 150 155 160 Lys Gly Ser ValSer Ile Met Thr Gly Gln Asp Glu Gly Ile Phe Ala 165 170 175 Trp Val ThrVal Asn Phe Leu Thr Gly Gln Leu His Gly His Arg Gln 180 185 190 Glu ThrVal Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr 195 200 205 PheLeu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr 210 215 220Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr His 225 230235 240 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly245 250 255 Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr Phe Arg Ser AlaCys 260 265 270 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly ValLys Tyr 275 280 285 Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly Phe GluPro Cys Tyr 290 295 300 Ala Glu Val Leu Arg Val Val Arg Gly Lys Leu HisGln Pro Glu Glu 305 310 315 320 Val Gln Arg Gly Ser Phe Tyr Ala Phe SerTyr Tyr Tyr Asp Arg Ala 325 330 335 Val Asp Thr Asp Met Ile Asp Tyr GluLys Gly Gly Ile Leu Lys Val 340 345 350 Glu Asp Phe Glu Arg Lys Ala ArgGlu Val Cys Asp Asn Leu Glu Asn 355 360 365 Phe Thr Ser Gly Ser Pro PheLeu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 Ala Leu Leu Lys Asp GlyPhe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395 400 Leu Thr Lys LysVal Asn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415 Thr Phe HisLeu Leu Gln Ser Leu Gly Ile Ser His 420 425 8 9365 DNA Homo sapiensmisc_feature (3409) n = adenine or guanine or cytosine or thymidine 8gcctctgcag gtgtgcgagc aggattgctt ctgcaacaaa agcctccacc cagccacatc 60ttgggaaaag aatggccact tcttggggca cagtcttttt catgctggtg gtatcctgtg 120tttgcagcgc tgtctcccac aggaaccagc agacttggtt tgagggtatc ttcctgtctt 180ccatgtgccc catcaatgtc agcgccagca ccttgtatgg aattatgttt gatgcaggga 240gcactggaac tcgaattcat gtttacacct ttgtgcagaa aatgccaggt aagtgcaact 300gggrccctta gtagagtctg taaatccaca ctttagcatc tcctcccaga aacaaatatg 360ctgagagttt attatgtgaa ttacagaatc tcacacctag tggatgtctt tcttcagaga 420actttggact acaattgaac atgtgggtta tttatttatt tttatttatt tgttttgttt 480ttatttttta actttttttt tgagacaagg tcttgctttg ttgcccggtc tgtagtgcag 540tggcatgatg acacatcact gcaaccttga cctcctgggc tcaagcagtc cttccacctc 600agccccctga gttgttgaga ctacaggctt gtgccaccat gcccagctca tttttaaatt 660tttttataga gacctgctca gactggcctc aaactcctag gctcaattga tcctcccacc 720tcagcctccc aaagtactgg gattataggt gtaagtcacc atgcttggcc agaacacatg 780gcttaattca atgtgaaatt agaagagagc tgggctgtct gtagtctgaa acccatgtgt 840tcaaaaagaa tagttataat ttgttcttcc tctttaaaca tgggatactc cagggatcca 900taatattcag aatatgggga gtggttttgg gagaaggatc acatgagaat ttcactgcca 960tccttggaca tgaggctagg aatccctgaa gattaacttt ttctgaattt gtcagtgttt 1020tttcctcagg tcacttatgg agcctgggga aaggtggagg agttaggtgt ccaccagaga 1080aatggtagca gaaatggacc ctcagaggtt gctctagtcc ttctttccag tactcctgca 1140agacattcct cacaactagg atcattgggg taacttcagg gaagtcatag gaaaacttac 1200agagacagag cccagcatct gaagcagcct aacttttggt aaccagctct ctcttctgtt 1260ttgttccatg racaaaatag gacagcttcc aattctagaa ggggaagttt ttgattctgt 1320gaagccagga ctttctgctt ttgtagatca acctaagcag ggtgctgaga ccgttcaagg 1380gctcttagag gtggccaaag actcaatccc ccgaagtcac tggaaaaaga ccccagtggt 1440cctaaaggca acagcaggac tacgcttact gccagaacac aaagccaagg ctctgctctt 1500tgaggtaaag gagatcttca ggaagtcacc tttcctggta ccaaagggca gtgttagcat 1560catggatgga tccgacgaag gtgggagagg tgttgatatg cgttccaggg ggagaggggc 1620aggatcagtg aaagatctaa ctaaaggaac tggggccagg aataaacaga aggaatgaga 1680tagcaggaaa tagaagacag ggagaaggga acatgtgctc tagacatgga atttagagag 1740gaaaaaaaaa aaacaaggtt ggggccagga aagagaaaaa atgctctggg atctaatcct 1800tgtctttctt tctttttagg catattagct tgggttactg tgaattttct gacaggtaat 1860acatcctcaa gtttatcttt agagcttaac tagcttttac atgcatagtc agaggagtaa 1920aagcctcttc tttcattctg tattgtttct tcttctttaa aaaaggaaaa gaggctgggt 1980gtggcagttc atgcctgtta attccagcgc tttgggaggc tgagttgggc agatcacttg 2040aggccaggag ttcaagacca gcctggccaa catggcgaaa ctccgtctct accaaaaata 2100caaaaatagc tgggcatggt ggtgtgtacc tgtagtccca gctactcagg aggctggaga 2160atcacttgaa cccaggaggc agaggttgca gtgagctgag agccgagatt gcgccactgc 2220actccaggct ggatgataga gcaagactct gtctccaaaa aggccttcca aaaaaaaaaa 2280aaacacctgc cttgaaggcc tctgctgcaa caagagtcct tccgagttga cattcacctg 2340cagccttggg gctggggagc agtggagtat atatggaata ccttcagtgt atgataagag 2400caagagagac aagtgttggg ctgcccagga tgtcgaggct atttagagct ggctctcatt 2460tgacaggtca gctgcatggc cacagacagg agactgtggg gaccttggac ctagggggag 2520cctccaccca aatcacgttc ctgccccagt ttgaggtgag tcatttaatg aagatctggt 2580tagaagtgca cttggcaggc gtatcatggt gccaagaaag aggcgcccca ttttcagcca 2640gcagctctac cacgcttagg cagagtcaag tcaattaata actaggtgaa tgttcccttg 2700ccatctcact gttcagaatc ccttcgtttc ctcaagccta gtgagattag ccccttaatc 2760tgtcttcatc tctgattttt tgctgggagg gacgggtggt ggtgtgaaca tcttcaggta 2820attacagatc ctgaatagct ttttgctttt tctgatttgc agaaaactct ggaacaaamt 2880cyatrgggct acctcacttc ctttgagatg tttaacagca cttataagct ctatacacat 2940aggtgaggac ggggacaggg aagaagaata tttmwtkttg tatgatksty ytamctktss 3000maagcwtkct caaatctstk aytkyatctg attmgcaaaa acaaagdctg tgccaattcc 3060ctaaggccta tcaactgaaa cccggwccac ttacaaagcc ggaggagcct aagaggcttc 3120tccattcttg gcctcaaaag cattaatata tgacttaaga gtcaaaagtt ttggstgggg 3180cagtggcttc atgcctgtaa tccctgcact ttgggaggcc gaggtgggtg ggtcacctga 3240ggtcaggcgt ttragaccag cctggcaaac atggtgaaac cccgtctyta ctaaaataca 3300aaaattagct ggatatgaca gcgcacacct gtaatcctag ctattcagga ggctgaggca 3360ggagaatcat ttgaaccctg gaggcggaga ttgcagtgag ccgagatcnc accmctgcac 3420ttcagccgga gcgacagagc aagactcagt ctcaaaaaaa aaaaaaaaaa gaatcaaaag 3480ctttctgtag ggagaggaca cttcaagaag gctcaggcaa agctccttgc cagctccttt 3540gagctggcct tcagaggttc agaatccagc ctggaatgtg atcccagttg gggctaggag 3600ctaagctaaa gagagctttt ctgggaatgg ttcctagwgt gggaccctag gaattgtcac 3660tgtctctggc ctttgaatga taactgtggg gaattcttac tgcatagcct tgatccaaac 3720tgtgcagaaa ttaccccttg ttgaccacag gagatgaata tgtcacagac agaacaaggt 3780tttcatcttt ccagagggac acaggaacaa tgttactttt gaaagaggta gctttaggct 3840agagaacttc aggaccagca tgaaattagt caatcctgta ttttacagtt acctgggatt 3900tggattgaaa gctgcaagac tagcaaccct gggagccctg gagacagaag gtttgtctgg 3960gtacctgtgc tgggggggga tggtgagggt gacacagata ctccgcttgc ttcttccctt 4020ccttgatagc cattctatgg aggaaaagat tatgttgaat tgggaggcaa atgttgtata 4080atggacctaa taatggcaaa ctccttttct agtttataag ttcagaagtt ttgatgtata 4140ttattagcca tttttagaat gaggtctact tgttcagggg taacagccta tgtctaggca 4200gctgaagtgt ctgcagaaat cccaggcttt acgaatacat tcagcaggag cttgctcaag 4260ccctgagctt tacattggag gcacaggaag cagagtctgt tctacatgca ggtggaacaa 4320cagagtaact ccattgatct cttcacaggt caggcagaac tgggttcagt cccagtgttg 4380tgatatgagg cragtaacct atctgtgccc ctttcctcac attaaatgag aatttgcatt 4440taaggcactt tgtacagtaa tctgttattg ggatgacatc tattttgcat ttcagagtat 4500acaaaacatc ttcaagtata tttaattgaa gcctctcagc aaccagtgag gaaggtagca 4560tagcatttct ttcctgtttt tataaagggg aaagttgctg takgaaggtt ykrgatctct 4620twragatgtg atraaagcca tggacccctc tgacaaaagc acatatgcat gaaaatttgc 4680ttctggtttc agggggttca ccaaccccac aaagcctatc tttgaaccct gagttaagga 4740ttcctgtcac aggatgttgt catggaatta atttcatagg attttaaggc ccagccccca 4800tggtgaytct tttccacctc actggcttct tgcttgcctt cctccctctc tctcacttac 4860ttacctctta ccttgtgccc tggattcttt cagggactga tgggcacact ttccggagtg 4920cctgtttacc gagatggttg gaagcagagt ggatctttgg gggtgtgaaa taccagtatg 4980gtggcaacca agaaggcaag tgatgttttt tcactggtta aagttacgtt tacaatggaa 5040gctctggaaa agtcccatgg gaaacttttt ccagaactca agagaagctt atcttgttgc 5100agggasttat tccaaagatc ttggcatgcc tccaaggact aatgtgaagt gacagtgaac 5160aaagcagctg tcattctgca tcagccaagt gtcatggacc cattagatac ctgcccttag 5220ccaagtgctg tggtgcacat ctattgtcct agctactcca aaggttgagg caagaggatc 5280acttgagccc atgagttcaa ggctatagtg cgcaatgcca ctgcactcca gcctgggcaa 5340cagggagacc ctacctctta caaattaatt aagaagcata ttctaagcct aggtctaatg 5400cagcagtgtg aaagcctgtt tagttaatgg ttagctattt aaattatagt aaaacttaaa 5460accaagacaa gaatgattca tcttcttata aaaggtatat acctgaatat caaggaatga 5520acctgaattc ccagtgaagg aagcaggcga gccctttagc tacttgctta caaatgctat 5580ggaatgtaat gctaggcagc agcacaaggt tggccatgat ctggtgaata cagattaggc 5640aggagagcgg ccatggagaa acagactggt gaggctgcag acgtttgctc atctttgttt 5700tgacgcctct tgtcccaagc ctcagccttc tcctgctttc ttgaccttcc tgctgttccc 5760tcattgtctc cagcagcctg cctcagagag tgtccccttc ccccagcgtc gttctcacct 5820tacccctgtg cacctttgcc tggcagggga ggtgggcttt gagccctgct atgccgaagt 5880gctgagggtg gtacgaggaa aacttcacca gccagaggag gtccagagag gttccttcta 5940tgctttctct tactattatg accgagctgt tgacacagac atgattggtg agttcacccc 6000aggtgtcagt ccagagagga aggtggatag ggctgtggtg gggaaggtca aggagaaaga 6060gcacttgagg tgctttgtcg gggtgattac ccacctcttt tctagtcact cgaacaaaag 6120ggtggaaatg acttagagtc ttttggaggt gagagatgac caaaacaact atatgaggtc 6180tttttttttt taacatgttt attgaggtat aattggcata caataagtgc cacatttaaa 6240gtatacaatt taagttttgt catgtataca cccatgaatc catccagcac attgaagata 6300ataaacatat ttcaccacaa aaagtttcct cctgtctctt tataactttt cttcttatca 6360caaaagcagt gtttttgcct aactgtgaaa gtatatgtac ctgatctgtc atggcctgag 6420agagatgaat taatttccta ttattgtggg ggttttgttg ttgttgttgt tttggttttt 6480tgtttgtttg tttgtttttt gagacagagt ctcactctgt tacccaggct ggagtgcaat 6540ggcatgatct aggctcactg caacctctgc ctcccgggtt caaccgattc tcctgcccca 6600gtctcctgag tagctgggat tacaggtgcc tgccaccaca cccggctaat ttttttttta 6660atagagacga ggtttcacca tgttggtcag gctggtcttg aactcctgac ctcgttatct 6720gccttcctcg gcctcccaaa gtgctgggat tacaggcatg agccaccaca cccggcctat 6780tgtgttttat gggtctgttt tttccattgt ggttaaatat acataacatg gaatagattg 6840taaataagta aattaggttg catagattac attatgtaca tgtgtatata atgaatgaat 6900gaatgaattt ccttatgctt ccttgaaggc gttttgatat cagataatct tctgttttat 6960ttcagattat gaaaaggggg gtattttaaa agttgaagat tttgaaagaa aagccaggga 7020agtgtgtgat aacttggaaa acttcacctc aggcagtcct ttcctgtgca tggatctcag 7080ctacatcaca gccctgttaa aggatggctt tggctttgca gacagcacag tcttacaggt 7140aagagacagg acaccagagt ctcataacag ccctcttttg tgggggttga gaaggagtaa 7200gagcttgttc agtaatcaga gtagctagaa gtgaaattat gaggtatttt tgtttgggct 7260atggacaagg tactgtgctg ggcaccatga atgtgggaaa ttatctcaat gcaatggtag 7320cctccgagtg tattaccagg caagctatcg cacaggtcac agaacagaaa gactagcagc 7380ccaaattaag atgccaagtc acatggttta tttatttatt tatttattta ttattatttt 7440tttgagacgg agtctygctc ttgttkccyr ggctggagtg cartggcryg atcwcrgctc 7500actgcarcct ycrcctcctg ggttcaagcg attctyctgc ctcagcctcc cragtagctg 7560ggattacagg crygcgccac cacgccyggc taattttttt gtatttttag tagagacggg 7620gtttcaccat gttggccagg ctrktctyra actyctgayc tcaggtgatc cacccrcctc 7680rgcctcccaa agtgctrgra ttayaggyrt gagccaccac kccyrgcctt ttttgktcgk 7740ttcttttttt ttchtttttt tttttttttt gagacagggt cttgctctgt cacccatgct 7800ggagtgcagt ggcatgatct cagttcactg caacctctgc ctcccgggtt caagtgaccc 7860tcccacctca gccctctgag tagctgggat tacaggtgtg tgccaccact cttgtctaat 7920ttttttgtag agacggggtt ttgccatgtt gcccaggctg gtcttgaact cctggcctca 7980agcaatccac ctgccttggc ctcccaaagt gccaggagta caggcatgag ccactgcgcc 8040tggccccatg tttggttatt attagtgctt aggaagaggc acttgcttac atagtaggag 8100ttgagaagct tggtttgttc tttcctaccc ctagatctat tctcacctcc tgaccatgct 8160ctttctgcca catctattat cattacaagt tgccttatct gaaattagtg aatcagaaaa 8220taaagcaggg gatactttgt gtagtttcaa cgttagggaa agttcagaat actgtctgtc 8280taaactatct ctctagaagg cctgatgggc cacaacctgg gccagaagca ttcagttcag 8340atatgagaat ggtgggtgta ggggcaatgg ccaatgggcc atggccggaa ggaaattgtt 8400acagagtagt gggaagcctg caaagactgg cttctgtccg ttttgccttg gtttgcccat 8460gtggatattc tttgccaata ttttctgccc aagagctgtg cttgctagag ttggaaactg 8520gatgaaaagg tgaagacttt ttttcttctc aacagctcac aaagaaagtg aacaacatag 8580agacgggctg ggccttgggg gccacctttc acctgttgca gtctctgggc atctcccatt 8640gaggccacgt acttccttgg agacctgcat ttgccaacac ctttttaagg ggaggagaga 8700gcacttagtt tctgaactag tctggggaca tcctggactt gagcctagag atttaggttt 8760aattaatttt acacatctaa tagtgaactg ctgcctaacc actcaagagt acacagctgg 8820caccagagca tcacagagag ccctgtgagc caaaaagtat agttttggaa cttaaccttg 8880gagtgagagc ccagggacag gtccctggaa accaaagaaa aatcgcattt caaccctttg 8940agtgcctcat tccactgaat atttaaattt tcctcttaaa tgggaaactg acttattgca 9000atcccaagac ccatcaatat cagtattttt ttcctcccta tacagggccc tgcccaccct 9060tatctgcacc cacctcccct gaaaaagaga gaaaaaaaaa aamccbggtt ttgctttccw 9120tgtwtaatyc amcgacmcaa aakgggacca tgtcaaaatc tgtwtgatcc tattytgggt 9180tascyccaat cagccagctg aragccttcc taanttttaw taggatgara gagtaccycc 9240taactgtgca taaattcagc cttaaaaaaa aaggcacccg ggctttgggg acatgtttgg 9300ganggggggg ntgcctcata tacccacctt tggtttaata acattttatc agcactttgg 9360gataa 9365 9 20 DNA Artificial Sequence Description of ArtificialSequence primer 9 gctacctcac ttcctttgag 20 10 21 DNA Artificial SequenceDescription of Artificial Sequence primer 10 ctggctggtg aagttttcct c 2111 21 DNA Artificial Sequence Description of Artificial Sequence primer11 gcaggtctcc aaggaagtac g 21 12 30 DNA Artificial Sequence Descriptionof Artificial Sequence primer 12 gtgagtgctc cctgcatcta acataattcc 30 1345 DNA Artificial Sequence Description of Artificial Sequence primer 13gatgcaggga gcactcacac tagtattcat gtttacacct ttgtg 45 14 44 DNAArtificial Sequence Description of Artificial Sequence primer 14gcgtagtcct gctgttgccc ctaggtacac tggggtcttt ttcc 44 15 31 DNA ArtificialSequence Description of Artificial Sequence primer 15 gcaacagcaggactacgctt actgccagaa c 31 16 48 DNA Artificial Sequence Description ofArtificial Sequence primer 16 cccaagcgaa tatgccttcg tcttgtccagtcatgatgct aacactgc 48 17 28 DNA Artificial Sequence Description ofArtificial Sequence primer 17 cgaaggcata ttcgcttggg ttactgtg 28 18 22DNA Artificial Sequence Description of Artificial Sequence primer 18cttccttcac tgggaattca gg 22 19 24 DNA Artificial Sequence Description ofArtificial Sequence primer 19 ctgtttaccg agatggttgg aagc 24 20 29 DNAArtificial Sequence Description of Artificial Sequence primer 20ttaaagcttg ggaaaagaat ggccacttc 29 21 29 DNA Artificial SequenceDescription of Artificial Sequence primer 21 agactcgagg tggctcaatgggagatgcc 29 22 58 DNA Artificial Sequence Description of ArtificialSequence primer 22 gcgctgtctc ccacagagga tcgcatcacc atcaccatcacaaccagcag acttggtt 58 23 58 DNA Artificial Sequence Description ofArtificial Sequence primer 23 aaccaagtct gctggttgtg atggtgatggtgatgcgatc ctctgtggga gacagcgc 58 24 1601 DNA Homo sapiens 24 gcgggctgccgcgcaagggt ggcgcgcgcg cgttttcctt gttcctggtc aacaaagaaa 60 tgtggagtgtcttggctgaa tcctcataca gacaagatca ttatggtgct gttaggttga 120 aaaagtgatataataaagga accaaggaga aaattcagaa ggaaagaaaa aattgcctct 180 gcaggtgtgcgagcaggatt gcttctgcaa caaaagcctc cacccagcca catcttggga 240 aaagaatggccacttcttgg ggcacagtct ttttcatgct ggtggtatcc tgtgtttgca 300 gcgctgtctcccacaggaac cagcagactt ggtttgaggg tatcttcctg tcttccatgt 360 gccccatcaatgtcagcgcc agcaccttgt atggaattat gtttgatgca gggagcactg 420 gaactcgaattcatgtttac acctttgtgc agaaaatgcc aggacagctt ccaattctag 480 aaggggaagtttttgattct gtgaagccag gactttctgc ttttgtagat caacctaagc 540 agggtgctgagaccgttcaa gggctcttag aggtggccaa agactcaatc ccccgaagtc 600 actggaaaaagaccccagtg gtcctaaagg caacagcagg actacgctta ctgccagaac 660 acaaagccaaggctctgctc tttgaggtaa aggagatctt caggaagtca cctttcctgg 720 taccaaagggcagtgttagc atcatggatg gatccgacga aggcatatta gcttgggtta 780 ctgtgaattttctgacaggt cagctgcatg gccacagaca ggagactgtg gggaccttgg 840 acctagggggagcctccacc caaatcacgt tcctgcccca gtttgagaaa actctggaac 900 aaactcctaggggctacctc acttcctttg agatgtttaa cagcacttat aagctctata 960 cacatagttacctgggattt ggattgaaag ctgcaagact agcaaccctg ggagccctgg 1020 agacagaagggactgatggg cacactttcc ggagtgcctg tttaccgaga tggttggaag 1080 cagagtggatctttgggggt gtgaaatacc agtatggtgg caaccaagaa ggggaggtgg 1140 gctttgagccctgctatgcc gaagtgctga gggtggtacg aggaaaactt caccagccag 1200 aggaggtccagagaggttcc ttctatgctt tctcttacta ttatgaccga gctgttgaca 1260 cagacatgattgattatgaa aaggggggta ttttaaaagt tgaagatttt gaaagaaaag 1320 ccagggaagtgtgtgataac ttggaaaact tcacctcagg cagtcctttc ctgtgcatgg 1380 atctcagctacatcacagcc ctgttaaagg atggctttgg ctttgcagac agcacagtct 1440 tacaggctgccgtactgagg tgatgggcca agctggagat atccccaaag cccatgttga 1500 caccctgtcctgcaagcgga tggactctgt gggctgcatc cctaagaata aagcagagtt 1560 caggtgtgacctctggcagc aaaaaaaaaa aaaaaaaaaa a 1601 25 405 PRT Homo sapiens 25 MetAla Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 7580 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 9095 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu Lys115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys AlaLeu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe LeuVal Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp GluGly Ile Leu Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly Gln LeuHis Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu Gly GlyAla Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys Thr LeuGlu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met Phe AsnSer Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly Phe GlyLeu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu Thr GluGly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro Arg TrpLeu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln Tyr GlyGly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 Ala GluVal Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr IleThr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr ValLeu Gln 385 390 395 400 Ala Ala Val Leu Arg 405 26 2762 DNA Homo sapiensCDS (148)..(1599) 26 gtggggtcgt atcccgcggg tggaggccgg ggtggcgccggccggggcgg gggagcccaa 60 aagaccggct gccgcctgct ccccggaaaa gggcactcgtctccgtgggt gtggcggagc 120 gcgcggtgca tggaatgggc tatgtga atg aaa aaa ggtatc cgt tat gaa act 174 Met Lys Lys Gly Ile Arg Tyr Glu Thr 1 5 tcc agaaaa acg agc tac att ttt cag cag ccg cag cac ggt cct tgg 222 Ser Arg LysThr Ser Tyr Ile Phe Gln Gln Pro Gln His Gly Pro Trp 10 15 20 25 caa acaagg atg aga aaa ata tcc aac cac ggg agc ctg cgg gtg gcg 270 Gln Thr ArgMet Arg Lys Ile Ser Asn His Gly Ser Leu Arg Val Ala 30 35 40 aag gtg gcatac ccc ctg ggg ctg tgt gtg ggc gtg ttc atc tat gtt 318 Lys Val Ala TyrPro Leu Gly Leu Cys Val Gly Val Phe Ile Tyr Val 45 50 55 gcc tac atc aagtgg cac cgg gcc acc gcc acc cag gcc ttc ttc agc 366 Ala Tyr Ile Lys TrpHis Arg Ala Thr Ala Thr Gln Ala Phe Phe Ser 60 65 70 atc acc agg gca gccccg ggg gcc cgg tgg ggt cag cag gcc cac agc 414 Ile Thr Arg Ala Ala ProGly Ala Arg Trp Gly Gln Gln Ala His Ser 75 80 85 ccc ctg ggg aca gct gcagac ggg cac gag gtc ttc tac ggg atc atg 462 Pro Leu Gly Thr Ala Ala AspGly His Glu Val Phe Tyr Gly Ile Met 90 95 100 105 ttt gat gca gga agcact ggc acc cga gta cac gtc ttc cag ttc acc 510 Phe Asp Ala Gly Ser ThrGly Thr Arg Val His Val Phe Gln Phe Thr 110 115 120 cgg ccc ccc aga gaaact ccc acg tta acc cac gaa acc ttc aaa gca 558 Arg Pro Pro Arg Glu ThrPro Thr Leu Thr His Glu Thr Phe Lys Ala 125 130 135 gtg aag cca ggt ctttct gcc tat gct gat gat gtt gaa aag agc gct 606 Val Lys Pro Gly Leu SerAla Tyr Ala Asp Asp Val Glu Lys Ser Ala 140 145 150 cag gga atc cgg gaacta ctg gat gtt gct aaa cag gac att ccg ttc 654 Gln Gly Ile Arg Glu LeuLeu Asp Val Ala Lys Gln Asp Ile Pro Phe 155 160 165 gac ttc tgg aag gccacc cct ctg gtc ctc aag gcc aca gct ggc tta 702 Asp Phe Trp Lys Ala ThrPro Leu Val Leu Lys Ala Thr Ala Gly Leu 170 175 180 185 cgc ctg tta cctgga gaa aag gcc cag aag tta ctg cag aag gtg aaa 750 Arg Leu Leu Pro GlyGlu Lys Ala Gln Lys Leu Leu Gln Lys Val Lys 190 195 200 gaa gta ttt aaagca tcg cct ttc ctt gta ggg gat gac tgt gtt tcc 798 Glu Val Phe Lys AlaSer Pro Phe Leu Val Gly Asp Asp Cys Val Ser 205 210 215 atc atg aac ggaaca gat gaa ggc gtt tcg gcg tgg atc acc atc aac 846 Ile Met Asn Gly ThrAsp Glu Gly Val Ser Ala Trp Ile Thr Ile Asn 220 225 230 ttc ctg aca ggcagc ttg aaa act cca gga ggg agc agc gtg ggc atg 894 Phe Leu Thr Gly SerLeu Lys Thr Pro Gly Gly Ser Ser Val Gly Met 235 240 245 ctg gac ttg ggcgga gga tcc act cag atc gcc ttc ctg cca cgc gtg 942 Leu Asp Leu Gly GlyGly Ser Thr Gln Ile Ala Phe Leu Pro Arg Val 250 255 260 265 gag ggc accctg cag gcc tcc cca ccc ggc tac ctg acg gca ctg cgg 990 Glu Gly Thr LeuGln Ala Ser Pro Pro Gly Tyr Leu Thr Ala Leu Arg 270 275 280 atg ttt aacagg acc tac aag ctc tat tcc tac agc tac ctc ggg ctc 1038 Met Phe Asn ArgThr Tyr Lys Leu Tyr Ser Tyr Ser Tyr Leu Gly Leu 285 290 295 ggg ctg atgtcg gca cgc ctg gcg atc ctg ggc ggc gtg gag ggg cag 1086 Gly Leu Met SerAla Arg Leu Ala Ile Leu Gly Gly Val Glu Gly Gln 300 305 310 cct gct aaggat gga aag gag ttg gtc agc cct tgc ttg tct ccc agt 1134 Pro Ala Lys AspGly Lys Glu Leu Val Ser Pro Cys Leu Ser Pro Ser 315 320 325 ttc aaa ggagag tgg gaa cac gca gaa gtc acg tac agg gtt tca ggg 1182 Phe Lys Gly GluTrp Glu His Ala Glu Val Thr Tyr Arg Val Ser Gly 330 335 340 345 cag aaagca gcg gca agc ctg cac gag ctg tgt gct gcc aga gtg tca 1230 Gln Lys AlaAla Ala Ser Leu His Glu Leu Cys Ala Ala Arg Val Ser 350 355 360 gag gtcctt caa aac aga gtg cac agg acg gag gaa gtg aag cat gtg 1278 Glu Val LeuGln Asn Arg Val His Arg Thr Glu Glu Val Lys His Val 365 370 375 gac ttctat gct ttc tcc tac tat tac gac ctt gca gct ggt gtg ggc 1326 Asp Phe TyrAla Phe Ser Tyr Tyr Tyr Asp Leu Ala Ala Gly Val Gly 380 385 390 ctc atagat gcg gag aag gga ggc agc ctg gtg gtg ggg gac ttc gag 1374 Leu Ile AspAla Glu Lys Gly Gly Ser Leu Val Val Gly Asp Phe Glu 395 400 405 atc gcagcc aag tac gtg tgt cgg acc ctg gag aca cag ccg cag agc 1422 Ile Ala AlaLys Tyr Val Cys Arg Thr Leu Glu Thr Gln Pro Gln Ser 410 415 420 425 agcccc ttc tca tgc atg gac ctc acc tac gtc agc ctg cta ctc cag 1470 Ser ProPhe Ser Cys Met Asp Leu Thr Tyr Val Ser Leu Leu Leu Gln 430 435 440 gagttc ggc ttt ccc agg agc aaa gtg ctg aag ctc act cgg aaa att 1518 Glu PheGly Phe Pro Arg Ser Lys Val Leu Lys Leu Thr Arg Lys Ile 445 450 455 gacaat gtt gag acc agc tgg gct ctg ggg gcc att ttt cat tac atc 1566 Asp AsnVal Glu Thr Ser Trp Ala Leu Gly Ala Ile Phe His Tyr Ile 460 465 470 gactcc ctg aac aga cag aag agt cca gcc tca tagtggccga gccatccctg 1619 AspSer Leu Asn Arg Gln Lys Ser Pro Ala Ser 475 480 tccccgtcag cagtgtctgtgtgtctgcat aaaccctcct gtcctggacg tgacttcatc 1679 ctgaggagcc acagcacaggccgtgctggc actttctgca cactggctct gggacttgca 1739 gaaggcctgg tgctgccctggcatcagcct cttccagtca catctggcca gagggctgtc 1799 tggacctggg ccctgctcaatgccacctgt ctgcctgggc tccaagtggg caggaccagg 1859 acagaaccac aggcacacactgagggggca gtgtggctcc ctgcctgtcc catccccatg 1919 ccccgtccgc ggggctgtggctgctgctgt gcatgtccct gcgatgggag tcttgtctcc 1979 cagcctgtca gtttcctccccagggcagag ctccccttcc tgcaagagtc tgggaggcgg 2039 tgcaggctgt cctggctgctctggggaagc cgagggacag ccataacacc cccgggacag 2099 taggtctggg cggcaccactgggaactctg gacttgagtg tgtttgctct tccttgggta 2159 tgaatgtgtg agttcacccagaggcctgct ctcctcacac attgtgtggt ttggggttaa 2219 tgatggaggg agacacctcttcatagacgg caggtgccca cctttcaggg agtctcccag 2279 catgggcgga tgccgggcatgagctgctgt aaactatttg tggctgtgct gcttgagtga 2339 cgtctctgtc gtgtgggtgccaagtgcttg tgtagaaact gtgttctgag cccccttttc 2399 tggacaccaa ctgtgtcctgtgaatgtatc gctactgtga gctgttcccg cctagccagg 2459 gccatgtctt aggtgcagctgtgccacggg tcagctgagc cacagtccca gaaccaagct 2519 ctcggtgtct cgggccaccatccgcccacc tcgggctgac cccacctcct ccatggacag 2579 tgtgagcccc gggccgtgcatcctgctcag tgtggcgtca gtgtcggggc tgagcccctt 2639 gagctgcttc agtgaatgtacagtgcccgg cacgagctga acctcatgtg ttccactccc 2699 aataaaaggt tgacaggggcttctccttca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2759 aaa 2762 27 484 PRTHomo sapiens 27 Met Lys Lys Gly Ile Arg Tyr Glu Thr Ser Arg Lys Thr SerTyr Ile 1 5 10 15 Phe Gln Gln Pro Gln His Gly Pro Trp Gln Thr Arg MetArg Lys Ile 20 25 30 Ser Asn His Gly Ser Leu Arg Val Ala Lys Val Ala TyrPro Leu Gly 35 40 45 Leu Cys Val Gly Val Phe Ile Tyr Val Ala Tyr Ile LysTrp His Arg 50 55 60 Ala Thr Ala Thr Gln Ala Phe Phe Ser Ile Thr Arg AlaAla Pro Gly 65 70 75 80 Ala Arg Trp Gly Gln Gln Ala His Ser Pro Leu GlyThr Ala Ala Asp 85 90 95 Gly His Glu Val Phe Tyr Gly Ile Met Phe Asp AlaGly Ser Thr Gly 100 105 110 Thr Arg Val His Val Phe Gln Phe Thr Arg ProPro Arg Glu Thr Pro 115 120 125 Thr Leu Thr His Glu Thr Phe Lys Ala ValLys Pro Gly Leu Ser Ala 130 135 140 Tyr Ala Asp Asp Val Glu Lys Ser AlaGln Gly Ile Arg Glu Leu Leu 145 150 155 160 Asp Val Ala Lys Gln Asp IlePro Phe Asp Phe Trp Lys Ala Thr Pro 165 170 175 Leu Val Leu Lys Ala ThrAla Gly Leu Arg Leu Leu Pro Gly Glu Lys 180 185 190 Ala Gln Lys Leu LeuGln Lys Val Lys Glu Val Phe Lys Ala Ser Pro 195 200 205 Phe Leu Val GlyAsp Asp Cys Val Ser Ile Met Asn Gly Thr Asp Glu 210 215 220 Gly Val SerAla Trp Ile Thr Ile Asn Phe Leu Thr Gly Ser Leu Lys 225 230 235 240 ThrPro Gly Gly Ser Ser Val Gly Met Leu Asp Leu Gly Gly Gly Ser 245 250 255Thr Gln Ile Ala Phe Leu Pro Arg Val Glu Gly Thr Leu Gln Ala Ser 260 265270 Pro Pro Gly Tyr Leu Thr Ala Leu Arg Met Phe Asn Arg Thr Tyr Lys 275280 285 Leu Tyr Ser Tyr Ser Tyr Leu Gly Leu Gly Leu Met Ser Ala Arg Leu290 295 300 Ala Ile Leu Gly Gly Val Glu Gly Gln Pro Ala Lys Asp Gly LysGlu 305 310 315 320 Leu Val Ser Pro Cys Leu Ser Pro Ser Phe Lys Gly GluTrp Glu His 325 330 335 Ala Glu Val Thr Tyr Arg Val Ser Gly Gln Lys AlaAla Ala Ser Leu 340 345 350 His Glu Leu Cys Ala Ala Arg Val Ser Glu ValLeu Gln Asn Arg Val 355 360 365 His Arg Thr Glu Glu Val Lys His Val AspPhe Tyr Ala Phe Ser Tyr 370 375 380 Tyr Tyr Asp Leu Ala Ala Gly Val GlyLeu Ile Asp Ala Glu Lys Gly 385 390 395 400 Gly Ser Leu Val Val Gly AspPhe Glu Ile Ala Ala Lys Tyr Val Cys 405 410 415 Arg Thr Leu Glu Thr GlnPro Gln Ser Ser Pro Phe Ser Cys Met Asp 420 425 430 Leu Thr Tyr Val SerLeu Leu Leu Gln Glu Phe Gly Phe Pro Arg Ser 435 440 445 Lys Val Leu LysLeu Thr Arg Lys Ile Asp Asn Val Glu Thr Ser Trp 450 455 460 Ala Leu GlyAla Ile Phe His Tyr Ile Asp Ser Leu Asn Arg Gln Lys 465 470 475 480 SerPro Ala Ser 28 27 DNA Artificial Sequence Description of ArtificialSequence primer 28 cgtatcccgc gggtggaggc cggggtg 27 29 27 DNA ArtificialSequence Description of Artificial Sequence primer 29 cttctgcaagtcccagagcc agtgtgc 27 30 21 DNA Artificial Sequence Description ofArtificial Sequence primer 30 ggagcccaaa agaccggctg c 21 31 21 DNAArtificial Sequence Description of Artificial Sequence primer 31tgaagtcacg tccaggacag g 21 32 36 DNA Artificial Sequence Description ofArtificial Sequence primer 32 cggaattcaa catgaaaaaa ggtaatccgt tatgaa 3633 26 DNA Artificial Sequence Description of Artificial Sequence primer33 tgtctagatg aggctggact cttctg 26 34 21 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide primer 34 atcctggacttgagcctaga g 21 35 21 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 35 ctgatattga tgggtcttgg g 21 36 21 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideprimer 36 ggatggaaag gagttggtca g 21 37 21 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide primer 37 gtccacatgcttcacttcct c 21 38 28 DNA Artificial Sequence Description of ArtificialSequence primer 38 cggatccctg tgctacgtcg ccctggac 28 39 32 DNAArtificial Sequence Description of Artificial Sequence primer 39cggaattcac tggcgcaggc ggtgatctcc tt 32 40 21 DNA Artificial SequenceDescription of Artificial Sequence primer 40 catcctgagg agccacagca c 2141 21 DNA Artificial Sequence Description of Artificial Sequence primer41 aggttcagct cgtgccgggc a 21 42 14747 DNA Homo sapiens misc_feature(13641) n = adenosine or guanine or cytosine or thymidine 42 aactagatacccttcggaaa aggtgctaag agcccacatc accctggttt agcttgttgt 60 acaggttattagaataggga aggaacacct tgggcccagg agtgagtgtt tcttctttta 120 caggtgtaactaaaaaagct agttggtcta acttgtgtgt ctttgtccta taagcctctg 180 caggtgtgcgagcaggattg cttctgcaac aaaagcctcc acccagccac atcttgggaa 240 aagaatggccacttcttggg gcacagtctt tttcatgctg gtggtatcct gtgtttgcag 300 cgctgtctcccacaggaacc agcagacttg gtttgagggt atcttcctgt cttccatgtg 360 ccccatcaatgtcagcgcca gcaccttgta tggaattatg tttgatgcag ggagcactgg 420 aactcgaattcatgtttaca cctttgtgca gaaaatgcca ggtaagtgca actgggrccc 480 ttagtagagtctgtaaatcc acactttagc atctcctccc agaaacaaat atgctgagag 540 tttattatgtgaattacaga atctcacacc tagtggatgt ctttcttcag agaactttgg 600 actacaattgaacatgtggg ttatttattt atttttattt atttgttttg tttttatttt 660 ttaactttttttttgagaca aggtcttgct ttgttgcccg gtctgtagtg cagtggcatg 720 atgacacatcactgcaacct tgacctcctg ggctcaagca gtccttccac ctcagccccc 780 tgagttgttgagactacagg cttgtgccac catgcccagc tcatttttaa atttttttat 840 agagacctgctcagactggc ctcaaactcc taggctcaat tgatcctccc acctcagcct 900 cccaaagtactgggattata ggtgtaagtc accatgcttg gccagaacac atggcttaat 960 tcaatgtgaaattagaagag agctgggctg tctgtagtct gaaacccatg tgttcaaaaa 1020 gaatagttataatttgttct tcctctttaa acatgggata ctccagggat ccataatatt 1080 cagaatatggggagtggttt tgggagaagg atcacatgag aatttcactg ccatccttgg 1140 acatgaggctaggaatccct gaagattaac tttttctgaa tttgtcagtg ttttttcctc 1200 aggtcacttatggagcctgg ggaaaggtgg aggagttagg tgtccaccag agaaatggta 1260 gcagaaatggaccctcagag gttgctctag tccttctttc cagtactcct gcaagacatt 1320 cctcacaactaggatcattg gggtaacttc agggaagtca taggaaaact tacagagaca 1380 gagcccagcatctgaagcag cctaactttt ggtaaccagc tctctcttct gttttgttcc 1440 atggacaaaataggacagct tccaattcta gaaggggaag tttttgattc tgtgaagcca 1500 ggactttctgcttttgtaga tcaacctaag caggtgagyt tcttatgatt tgatgtttag 1560 attctcratgccttgataac ttgaccacac cactgctgtt aaatatttca tgctattcac 1620 taatgagattgagatcatgt gtgagatcag ctttctctcc tcagctaatc tcctggataa 1680 aattaattacatttcccacg ctcatgaggg ctgggtggaa gaaggctaga aaattggttg 1740 tggtaatgcaaggatgacac tagcttttaa gagattactc cctatgtact tactgtttaa 1800 aattttgagttgaagccatc agttgtattt gttcatgtaa gcgcagaaga taaacatgaa 1860 gttctgtagggtatgatgta taaacatggc agttgaaggc atggtgagca gtgaattcca 1920 tttcagagtgccttgatgca ggatggaacc atatctatac ttgagaaatt aggcaacaac 1980 ccatgatgacaaaaatcttt ggtggtagaa gatgaataga gagtttgtgg cgactatatt 2040 tcacatttgagcatctacaa catactacat cctatgtgtc tgaggccatg ttgaaataaa 2100 atagaataatgcagaatgtt aaacttgggg agcaaatgta aaggtattta ttgttcattg 2160 tcacaagaataatcattacc caaatgcttg atgttaaaaa tgatttgagt ttggagaact 2220 gagaggatatatggatatat atacattttc ttctttttct tttttttttt ttttttgacg 2280 gagtatcactctgttaccca ggctggagtg cagtggcctg atctcggctt actacaacct 2340 ctgcctcgcgggttcaagca attcttctgc ctcagcctcc cgagtagctg ggactacagg 2400 catgtgccaccatgcccggc taatttttgt atttttagta gagacggggt ttcactatga 2460 tggycaggctggtcttgaac tcctgacctt gtgatccrcc catgtcagcc tcccaaagtt 2520 ctgggattacaggtgtgaga caccactgcg cctggcctgg attctccttc aaagcggccc 2580 acttctctaggtttctcctg ctacagagca gagagaggtt ggagccctat gccacctccc 2640 tcttcttgctcccasaaagt atgttgacag aatagaccag tgccagccac taaatggatc 2700 gttcatcagatgaacgggtt atctcttttg aagggtgctg agaccgttca agggctctta 2760 gaggtggccaaagactcaat cccccgaagt cactggaaaa agaccccagt ggtcctaaag 2820 gcaacagcaggactacgctt actgccagaa cacaaagcca aggctctgct ctttgaggta 2880 agttttaaaactgcatcttg gatcattctg cccttttccc tatatgaata ctttatgagt 2940 ttttagccttttggaatgtg accactacct tcagtattcc accactgcca aagcattgtg 3000 atgactctgaccacttgtta tagctagttg tttacatttt taactatcac tcttaaatgt 3060 atagagcttttcctaaaaaa tttaaagtac tttctatcta ggatattctt cattcttttt 3120 atttttatttttattttttg agatggagtc tcactctgtc acccaggctg gagtgcagtg 3180 gcgccatcttggctcactgc aagctccacc tcccaggtta atgccattct cctacctcag 3240 cctcccaggtagctgggatt acaggtgccc accaccatgc taattttttt gtatttttag 3300 tagagatggggtttcatcat gttggccagg ccggtctcga actcctgacc tcaggtgatc 3360 cacctgcctcggcctccaaa agtgctggga tgagaggtat gagccaccat gcccggctgc 3420 cactggaaggttttgagcca aggaaacaca tgatttgacg tccattttta aagcaccgtt 3480 ctggctgctgttttgagaac aaactgtagg atgggggaga aaagtggaga aggcaagagc 3540 agaagctctcttgctgtgct tcatcctgtt cacttatctg tctttaaaag tttgtttata 3600 tataacttaactggtttttc ctggttgaaa caggatccag aggtcattat gtggtatgtt 3660 tcatccatcagaaatgagaa cagcaaacag actagcgttc ttcctatcct attcggagaa 3720 ttcttttttctttcccttga taattatata aacataagac attaacaccc atatcaatgg 3780 tctgtattatggagaggcaa gatgtttttg caagatcgtc attcccatct cctgaaacag 3840 gattccttaagtctagatct ggagtctccc catgctctag agaccctagc catgtgccag 3900 aatctgacaggagctggtag cagagtacct cagcccttag gtgtgaactt caccacagca 3960 ggttcatgtcagctcatcta aggaatggag tgggaaggct cctcctagtt tccagtgtat 4020 gtccaggtagagtttatcag gtttaaaagt tacaggatcc tgatttgagc tggcagggtt 4080 aaatatatgcctttccatag gtccagttca cctatacatt tagatggttt ggtttagctt 4140 tacttaaagtcaaaggaatc tctgtgtttg tgtttttgca agaaactggt aatggcttgc 4200 ctagtttcttctctagtttc ttagggcaaa ggaatgagtt ttgccaaaat tttatctagg 4260 aaaaatggagtagttttcta agtcttacag aacactgtca aaatatggaa atctatttta 4320 ttgccttagggaattctttt tttttttttt cctttttctc taggtaaagg agatcttcag 4380 gaagtcacctttcctggtac caaagggcag tgttagcatc atggatggat ccgacgaagg 4440 tgggagaggtgttgatatgc gttccagggg gagaggggca ggatcagtga aagatctaac 4500 taaaggaactggggccagga ataaacagaa ggaatgagat agcaggaaat agaagacagg 4560 gagaagggaacatgtgctct agacatggaa tttagagagg aaaaaaaaaa aacaaggttg 4620 gggccaggaaagagaaaaaa tgctctggga tctaatcctt gtctttcttt ctttttaggc 4680 atattagcttgggttactgt gaattttctg acaggtaata catcctcaag tttatcttta 4740 gagcttaactagcttttaca tgcatagtca gaggagtaaa agcctcttct ttcattctgt 4800 attgtttcttcttctttaaa aaaggaaaag aggctgggtg tggcagttca tgcctgttaa 4860 ttccagcgctttgggaggct gagttgggca gatcacttga ggccaggagt tcaagaccag 4920 cctggccaacatggcgaaac tccgtctcta ccaaaaatac aaaaatagct gggcatggtg 4980 gtgtgtacctgtagtcccag ctactcagga ggctggagaa tcacttgaac ccaggaggca 5040 gaggttgcagtgagctgaga gccgagattg cgccactgca ctccaggctg gatgatagag 5100 caagactctgtctccaaaaa ggccttccaa aaaaaaaaaa aacacctgcc ttgaaggcct 5160 ctgctgcaacaagagtcctt ccgagttgac attcacctgc agccttgggg ctggggagca 5220 gtggagtatatatggaatac cttcagtgta tgataagagc aagagagaca agtgttgggc 5280 tgcccaggatgtcgaggcta tttagagctg gctctcattt gacaggtcag ctgcatggcc 5340 acagacaggagactgtgggg accttggacc tagggggagc ctccacccaa atcacgttcc 5400 tgccccagtttgaggtgagt catttaatga agatctggtt agaagtgcac ttggcaggcg 5460 tatcatggtgccaagaaaga ggcgccccat tttcagccag cagctctacc acgcttaggc 5520 agagtcaagtcaattaataa ctaggtgaat gttcccttgc catctcactg ttcagaatcc 5580 cttcgtttcctcaagcctag tgagattagc cccttaatct gtcttcatct ctgatttttt 5640 gctgggagggacgggtggtg gtgtgaacat cttcaggtaa ttacagatcc tgaatagtct 5700 ttttgctttttctgatttgc agaaaactct ggaacaaact cctaggggct acctcacttc 5760 ctttgagatgtttaacagca cttataagct ctatacacat aggtgaggac ggggacaggg 5820 aagaagaatatttcatgttg tatgattctc ctaactttcc aaagcattct caaatctgtt 5880 attgtatctgattagcaaaa acaaagtctg tgccaattcc ctaaggccta tcaactgaaa 5940 cccggtccacttacaaagcc ggaggagcct aagaggcttc tccattcttg gcctcaaaag 6000 cattaatatatgacttaaga gtcaaaagtt ttcggctggg tgcagtggct tcatgcctgt 6060 aatccctgcactttgggagg ccgaggtggg tgggtcacct gaggtcaggc gtttgagacc 6120 agcctggcaaacatggtgaa accccgtctc tactaaaata caaaaattag ctggatatga 6180 cagcgcacacctgtaatcct agctattcag gaggctgagg caggagaatc atttgaaccc 6240 tggaggcggagattgcagtg agccgagatc acaccactgc acttcagccg gagcgacaga 6300 gcaagactcagtctcaaaaa aaaaaaaaaa aagaatcaaa agctttctgt agggagagga 6360 cacttcaagaaggctcaggc aaagctcctt gccagctcct ttgagctggc cttcagaggt 6420 tcagaatccagcctggaatg tgatcccagt tggggctagg agctaagcta aagagagctt 6480 ttctgggaatggttcctagt gtgggaccct aggaattgtc actgtctctg gcctttgaat 6540 gataactgtggggaattctt actgcatagc cttgatccaa actgtgcaga aattacccct 6600 tgttgaccacaggagatgaa tatgtcacag acagaacaag gttttcatct ttccagaggg 6660 acacaggaacaatgttactt ttgaaagagg tagctttagg ctagagaact tcaggaccag 6720 catgaaattagtcaatcctg tattttacag ttacctggga tttggattga aagctgcaag 6780 actagcaaccctgggagccc tggagacaga aggtttgtct gggtacctgt gctggggggg 6840 gatggtgagggtgacacaga tactccgctt gcttcttccc ttccttgata gccattctat 6900 ggaggaaaagattatgttga attgggaggc aaatgttgta taatggacct aataatggca 6960 aactccttttctagtttata agttcagaag ttttgatgta tattattagc catttttaga 7020 atgaggtctacttgttcagg ggtaacagcc tatgtctagg cagctgaagt gtctgcagaa 7080 atcccaggctttacgaatac attcagcagg agcttgctca agccctgagc tttacattgg 7140 aggcacaggaagcagagtct gttctacatg caggtggaac aacagagtaa ctccattgat 7200 ctcttcacaggtcaggcaga actgggttca gtcccagtgt tgtgatatga ggcgagtaac 7260 ctatctgtgcccctttcctc acattaaatg agaatttgca tttaaggcac tttgtacagt 7320 aatctgttattgggatgaca tctattttgc atttcagagt atacaaaaca tcttcaagta 7380 tatttaattgaagcctctca gcaaccagtg aggaaggtag catagcattt ctttcctgtt 7440 tttataaaggtggaaagttg ctgtattgaa ggttttggat ctctttgaga tgtgatgaaa 7500 gccatggacccctctgacaa aagcacatat gcatgaaaat ttgcttctgg tttcaggggg 7560 ttcaccaaccccacaaagcc tatctttgaa ccctgagtta aggattcctg tcacaggatg 7620 ttgtcatggaattaatttca taggatttta aggcccagcc cccatggtga ttcttttcca 7680 cctcactggcttcttgcttg ccttcctccc tctctctcac ttacttacct cttaccttgt 7740 gccctggattctttcaggga ctgatgggca cactttccgg agtgcctgtt taccgagatg 7800 gttggaagcagagtggatct ttgggggtgt gaaataccag tatggtggca accaagaagg 7860 caagtgatgttttttcactg gttaaagtta cgtttacaat ggaagctctg gaaaagtccc 7920 atgggaaactttttccagaa ctcaagagaa gcttatcttg ttgcagggac ttattccaaa 7980 gatcttggcatgcctccaag gactaatgtg aagtgacagt gaacaaagca gctgtcattc 8040 tgcatcagccaagtgtcatg gacccattag atacctgccc ttagccaagt gctgtggtgc 8100 acatctattgtcctagctac tccaaaggtt gaggcaagag gatcacttga gcccatgagt 8160 tcaaggctatagtgcgcaat gccactgcac tccagcctgg gcaacaggga gaccctacct 8220 cttacaaattaattaagaag catattctaa gcctaggtct aatgcagcag tgtgaaagcc 8280 tgtttagttaatggttagct atttaaatta tagtaaaact taaaaccaag acaagaatga 8340 ttcatcttcttataaaaggt atatacctga atatcaagga atgaacctga attcccagtg 8400 aaggaagcaggcgagccctt tagctacttg cttacaaatg ctatggaatg taatgctagg 8460 cagcagcacaaggttggcca tgatctggtg aatacagatt aggcaggaga gcggccatgg 8520 agaaacagactggtgaggct gcagacgttt gctcatcttt gttttgacgc ctcttgtccc 8580 aagcctcagccttctcctgc tttccttgac cttcctgctg ttccctcatt gtctccagca 8640 gcctgcctcagagagtgtcc ccttccccca gcgtcgttct caccttaccc ctgtgcacct 8700 ttgcctggcaggggaggtgg gctttgagcc ctgctatgcc gaagtgctga gggtggtacg 8760 aggaaaacttcaccagccag aggaggtcca gagaggttcc ttctatgctt tctcttacta 8820 ttatgaccgagctgttgaca cagacatgat tggtgagttc accccaggtg tcagtccaga 8880 gaggaaggtggatagggctg tggtggggaa ggtcaaggag aaagagcact tgaggtgctt 8940 tgtcggggtgattacccacc tcttttctag tcactcgaac aaaagggtgg aaatgactta 9000 gagtcttttggaggtgagag atgaccaaaa caactatatg aggtcttttt ttttttaaca 9060 tgtttattgaggtataattg gcatacaata agtgccacat ttaaagtata caatttaagt 9120 tttgtcatgtatacacccat gaatccatcc agcacattga agataataaa catatttcac 9180 cacaaaaagtttcctcctgt ctctttataa cttttcttct tatcacaaaa gcagtgtttt 9240 tgcctaactgtgaaagtata tgtacctgat ctgtcatggc ctgagagaga tgaattaatt 9300 tcctattattgtgggggttt tgttgttgtt gttgttttgg ttttttgttt gtttgtttgt 9360 tttttgagacagagtctcac tctgttaccc aggctggagt gcaatggcat gatctaggct 9420 cactgcaacctctgcctccc gggttcaacc gattctcctg ccccagtctc ctgagtagct 9480 gggattacaggtgcctgcca ccacacccgg ctaatttttt ttttaataga gacgaggttt 9540 caccatgttggtcaggctgg tcttgaactc ctgacctcgt tatctgcctt cctcggcctc 9600 ccaaagtgctgggattacag gcatgagcca ccacacccgg cctattgtgt tttatgggtc 9660 tgttttttccattgtggtta aatatacata acatggaata gattgtaaat aagtaaatta 9720 ggttgcatagattacattat gtacatgtgt atataatgaa tgaatgaatg aatttcctta 9780 tgcttccttgaaggcgtttt gatatcagat aatcttctgt tttatttcag attatgaaaa 9840 ggggggtattttaaaagttg aagattttga aagaaaagcc agggaagtgc aagttcttca 9900 gaagttgcggtcttacagag gcagtcagtg cagtttagtc tttgagatgc ctaagcatta 9960 accaaagggagacttcwgct ttgcttattc ttcttctccc cctacctttt ttttttgaga 10020 cagagtcttgctctgtcacc caggctggag tgcagtggtg agatctcggc tcactgcaac 10080 ctctgccttccaggttcaag cgattctcct gcctcagcct cccgagtagc tgggattaca 10140 gcggtacaccaccacgccgt gctaattttt gtatttttag tagagatggg gtttcaccac 10200 gttggccaggctgatctcaa actcctgacc tcaggtgatc cacccgcctc agcctcccaa 10260 agtgctaggattacaggcgt gagccaccgc gcctggcccc tattccactt ctttctaaga 10320 gaaaatcctacacctctcag ttagttgcaa acttgagctc cactgttwac tctctctttc 10380 agtgtgtgataacttggaaa acttcacctc aggcagtcct ttcctgtgca tggatctcag 10440 ctacatcacagccctgttaa aggatggctt tggctttgca gacagcacag tcttacaggt 10500 aagagacaggacaccagagt ctcataacag ccctcttttg tgggggttga gaaggagtaa 10560 gagcttgttcagtaatcaga gtagctagaa gtgaaattat gaggtatttt tgtttgggct 10620 atggacaaggtactgtgctg ggcaccatga atgtgggaaa ttatctcaat gcaatggtag 10680 cctccgagtgtattaccagg caagctatcg cacaggtcac agaacagaaa gactagcagc 10740 ccaaattaagatgccaagtc acatggttta tttatttatt tatttattta ttattatttt 10800 tttgagacggagtctygctc ttgttkccyr ggctggagtg cartggcryg atcwcrgctc 10860 actgcarcctycrcctcctg ggttcaagcg attctyctgc ctcagcctcc cragtagctg 10920 ggattacaggcrygcgccac cacgccyggc taattttttt gtatttttag tagagacggg 10980 gtttcaccatgttggccagg ctrktctyra actyctgayc tcaggtgatc cacccrcctc 11040 rgcctcccaaagtgctrgra ttayaggyrt gagccaccac kccyrgcctt ttttgktcgk 11100 ttctttttttttchtttttt tttttttttt gagacagggt cttgctctgt cacccatgct 11160 ggagtgcagtggcatgatct cagttcactg caacctctgc ctcccgggtt caagtgaccc 11220 tcccacctcagccctctgag tagctgggat tacaggtgtg tgccaccact cttgtctaat 11280 ttttttgtagagacggggtt ttgccatgtt gcccaggctg gtcttgaact cctggcctca 11340 agcaatccacctgccttggc ctcccaaagt gccaggagta caggcatgag ccactgcgcc 11400 tggccccatgtttggttatt attagtgctt aggaagaggc acttgcttac atagtaggag 11460 ttgagaagcttggtttgttc tttcctaccc ctagatctat tctcacctcc tgaccatgct 11520 ctttctgccacatctattat cattacaagt tgccttatct gaaattagtg aatcagaaaa 11580 taaagcaggggatactttgt gtagtttcaa cgttagggaa agttcagaat actgtctgtc 11640 taaactatctctctagaagg cctgatgggc cacaacctgg gccagaagca ttcagttcag 11700 atatgagaatggtgggtgta ggggcaatgg ccaatgggcc atggccggaa ggaaattgtt 11760 acagagtagtgggaagcctg caaagactgg cttctgtccg ttttgccttg gtttgcccat 11820 gtggatattctttgccaata ttttctgccc aagagctgtg cttgctagag ttggaaactg 11880 gatgaaaaggtgaagacttt ttttcttctc aacagctcac aaagaaagtg aacaacatag 11940 agacgggctgggccttgggg gccacctttc acctgttgca gtctctgggc atctcccatt 12000 gaggccacgtacttccttgg agacctgcat ttgccaacac ctttttaagg ggaggagaga 12060 gcacttagtttctgaactag tctggggaca tcctggactt gagcctagag atttaggttt 12120 aattaattttacacatctaa tgtgaactgc tgcctaacca ctcaagagta cacagctggc 12180 accagagcatcacagagagc cctgtgagcc aaaaagtata gttttggaac ttaaccttgg 12240 agtgagagcccagggacagg tccctggaaa ccaaagaaaa atcgcatttc aaccctttga 12300 gtgcctcattccactgaata tttaaatttt cctcttaaat ggtaaactga cttattgcaa 12360 tcccaagacccatcaatatc agtatttttt tcctccctat acagtgccct gcccaccctt 12420 atctgcacccacctcccctg aaaaagagag aaaaaaaaaa acctggtttt gctttccatg 12480 tataattcaacaacgcaaaa tggtaccatg tcagaatctg tatgatccta ttctgtgtta 12540 gctccaatcagccagctgag agccatccta aatattaata ggatgagaga gtaactccta 12600 actgtgcataaattacagcc ttaagaaaga aggccacccg gtctctgggg acatgttttg 12660 ggagggtgtggctgcctcat atagcctacc tttgctttaa tcagcatttt atcagtcaac 12720 tctgggattaatgaacatat ccgactttat gggtatgtgt atattcagtg ccagtaccac 12780 ctcccaggctaatgtgttaa cagtgttttc tatgattcta aacttgtttt ctttgtattt 12840 ctaagaaatacaactaggcc ctaacttttg gtctgtggtc agaggtcagt gtctgtgcta 12900 ttccaggatctattatttca gtttgccctt ttattctctc cgtttgtaac agtaccttcc 12960 tgtcctggtccctgatcact ttagctaata tcctttgaat tatttatcta gttcagagtt 13020 tccaataccgttcctgacaa aaccccactt ggatttggtt ggctctctca gcccacatca 13080 gtacagtggaccaacatttc cagggagaac catatttaat gtccctcatg ctctcttttt 13140 gaatccaagtgctcagagct cttgactgtg aattatttac cccaaattca ttatcttgca 13200 cttttttttgagatggagtc tcgctctgtt gctcaggcta gagtggagtg gcgcaatctc 13260 agctcactgcaacctccgcc cccaggttca agcgawtctt gwtctcagcc tcctgaagag 13320 ctgggattacaggtgtgagc cactgcaccc atcagaaaaa acatatcttt aaatggaagt 13380 tctcagggatgaaactagtc aagcagcaaa aggagcttaa acatcttcat cattagaaat 13440 ccctattcattttttttttc aagagactgg tcttaactct gtcacccagt ctgagtaaca 13500 atgatgtgatcatagcccac tgcagcctca aactcctagg cttaaattat cctcttgcct 13560 cagkctcctgaatagctagg actacaggca tgtgccacca cactccctat tcactatttt 13620 taagccctttttatacatag ntattgctta gatgtgggcc gtaacaccta aaagcctagc 13680 aagtactcttctgcttgtaa cccttgggaa ggcaaacaca taaagataat acattttaca 13740 gccaagaaaattaattcaaa gaaattaaga gctgtgggcc agccgcagtg gctcacgcct 13800 gtaatcccagcactttggga ggccgaggca ggcagatcac gaggtcagga gttcaagacc 13860 agcttggccaacatggtgaa accccatctc tactaaaaat aagaaaatta gccgggtgtg 13920 gtggtgggcgcctgtaatcc cagctacttg ggaggctgag gcaggataat cacttgaacc 13980 tgggaggcagaggctgcagt gagtggaaat tgtgccactg cactccatcc tgggcgataa 14040 gaccaagactctgtttcaaa aaaaagcaaa agaaattaag agctgtttgc aaagtcattc 14100 ctctgagcaacctcttactt tgagtggaga cactgcattt gtggcctgca agccccacgt 14160 cctgagcaacacaagcgggg aggcactggt agaatagagc ctttttagta agtctgtagc 14220 agccagaagagcagtgttgt gacgctgtct ttctgtttca taacctgtga ggtggctcac 14280 ggaacctgaagggcagagga aagattaaat ccctaatcca ataataaggt tttcttccat 14340 ctcctagcaagccagatata ttttatgtcc actccctctt gctactgtgg gccctgatct 14400 tgcctaactatatgacctct ggatacttcc ccatagtacc tgaatcctta cctaagtcct 14460 tcccattgaaggctgccgta ctgaggtgat gggccaagct ggagatatcc ccaaagccca 14520 tgttgacaccctgtcctgca agcggatgga ctctgtgggc tgcatcccta agaataaagc 14580 agagttcaggtgtgacctct ggcagcaaag tgagaaggga gtggccctgc tctgtatcgt 14640 cttacccaatgagcgccacc cgaggcctga cgtactcagc agcatgttcc aacccaagag 14700 gaaacagaactcggcttttg gcatccaccc tgggctaccc cttgggg 14747 43 14 PRT ArtificialSequence Description of Artificial Sequence peptide 43 Glu Val Ala LysAsp Ser Ile Pro Arg Ser His Trp Lys Lys 1 5 10 44 14 PRT ArtificialSequence Description of Artificial Sequence peptide 44 Thr Arg Pro ProArg Glu Thr Pro Thr Leu Thr His Glu Thr 1 5 10 45 1498 DNA Homo sapiens45 atgaaaaaag gtatccgtta tgaaacttcc agaaaaacga gctacatttt tcagcagccg 60cagcacggtc cttggcaaac aaggatgaga aaaatatcca accacgggag cctgcgggtg 120gcgaaggtgg cataccccct ggggctgtgt gtgggcgtgt tcatctatgt tgcctacatc 180aagtggcacc gggccaccgc cacccaggcc ttcttcagca tcaccagggc agccccgggg 240gcccggtggg gtcagcaggc ccacagcccc ctggggacag ctgcagacgg gcacgaggtc 300ttctacggga tcatgtttga tgcaggaagc actggcaccc gagtacacgt cttccagttc 360acccggcccc ccagagaaac tcccacgtta acccacgaaa ccttcaaagc agtgaagcca 420ggtctttctg cctatgctga tgatgttgaa aagagcgctc agggaatccg ggaactactg 480gatgttgcta aacaggacat tccgttcgac ttctggaagg ccacccctct ggtcctcaag 540gccacagctg gcttacgcct gttacctgga gaaaaggccc agaagttact gcagaaggtg 600aaagaagtat ttaaagcatc gcctttcctt gtaggggatg actgtgtttc catcatgaac 660ggaacagatg aaggcgtttc ggcgtggatc accatcaact tcctgacagg cagcttgaaa 720actccaggag ggagcagcgt gggcatgctg gacttgggcg gaggatccac tcagatcgcc 780ttcctgccac gcgtggaggg caccctgcag gcctccccac ccggctacct gacggcactg 840cggatgttta acaggaccta caagctctat tcctacagct acctcgggct cgggctgatg 900tcggcacgcc tggcgatcct gggcggcgtg gaggggcagc ctgctaagga tggaaaggag 960ttggtcagcc cttgcttgtc tcccagtttc aaaggagagt gggaacacgc agaagtcacg 1020tacagggttt cagggcagaa agcagcggca agcctgcacg agctgtgtgc tgccagagtg 1080tcagaggtcc ttcaaaacag agtgcacagg acggaggaag tgaagcatgt ggacttctat 1140gctttctcct actattacga ccttgcagct ggtgtgggcc tcatagatgc ggagaaggga 1200ggcagcctgg tggtggggga cttcgagatc gcagccaagt acggtgggag tcacctggag 1260cgtgaaggga cgtgtctcat ccccagtgtg tcggaccctg gagacacagc cgcagagcag 1320ccccttctca tgcatggacc tcacctacgt cagcctgcta ctccaggagt tcggctttcc 1380caggagcaaa gtgctgaagc tcactcggaa aattgacaat gttgagacca gctgggctct 1440gggggccatt tttcattaca tcgactccct gaacagacag aagagtccag cctcatag 1498 462371 DNA Homo sapiens 46 gtggggtcgt atcccgcggg tggaggccgg ggtggcgccggccggggcgg gggagcccaa 60 aagaccggct gccgcctgct ccccggaaaa gggcactcgtctccgtgggt gtggcggagc 120 gcgcggtgca tgaaactccc acgttaaccc acgaaaccttcaaagcagtg aagccaggtc 180 tttctgccta tgctgatgat gttgaaaaga gcgctcagggaatccgggaa ctactggatg 240 ttgctaaaca ggacattccg ttcgacttct ggaaggccacccctctggtc ctcaaggcca 300 cagctggctt acgcctgtta cctggagaaa aggcccagaagttactgcag aaggtgaaag 360 aagtatttaa agcatcgcct ttccttgtag gggatgactgtgtttccatc atgaacggaa 420 cagatgaagg cgtttcggcg tggatcacca tcaacttcctgacaggcagc ttgaaaactc 480 caggagggag cagcgtgggc atgctggact tgggcggaggatccactcag atcgccttcc 540 tgccacgcgt ggagggcacc ctgcaggcct ccccacccggctacctgacg gcactgcgga 600 tgtttaacag gacctacaag ctctattcct acagctacctcgggctcggg ctgatgtcgg 660 cacgcctggc gatcctgggc ggcgtggagg ggcagcctgctaaggatgga aaggagttgg 720 tcagcccttg cttgtctccc agtttcaaag gagagtgggaacacgcagaa gtcacgtaca 780 gggtttcagg gcagaaagca gcggcaagcc tgcacgagctgtgtgctgcc agagtgtcag 840 aggtccttca aaacagagtg cacaggacgg aggaagtgaagcatgtggac ttctatgctt 900 tctcctacta ttacgacctt gcagctggtg tgggcctcatagatgcggag aagggaggca 960 gcctggtggt gggggacttc gagatcgcag ccaagtacgtgtgtcggacc ctggagacac 1020 agccgcagag cagccccttc tcatgcatgg acctcacctacgtcagcctg ctactccagg 1080 agttcggctt tcccaggagc aaagtgctga agctcactcggaaaattgac aatgttgaga 1140 ccagctgggc tctgggggcc atttttcatt acatcgactccctgaacaga cagaagagtc 1200 cagcctcata gtggccgagc catccctgtc cccgtcagcagtgtctgtgt gtctgcataa 1260 accctcctgt cctggacgtg acttcatcct gaggagccacagcacaggcc gtgctggcac 1320 tttctgcaca ctggctctgg gacttgcaga aggcctggtgctgccctggc atcagcctct 1380 tccagtcaca tctggccaga gggctgtctg gacctgggccctgctcaatg ccacctgtct 1440 gcctgggctc caagtgggca ggaccaggac agaaccacaggcacacactg agggggcagt 1500 gtggctccct gcctgtccca tccccatgcc ccgtccgcggggctgtggct gctgctgtgc 1560 atgtccctgc gatgggagtc ttgtctccca gcctgtcagtttcctcccca gggcagagct 1620 ccccttcctg caagagtctg ggaggcggtg caggctgtcctggctgctct ggggaagccg 1680 agggacagcc ataacacccc cgggacagta ggtctgggcggcaccactgg gaactctgga 1740 cttgagtgtg tttgctcttc cttgggtatg aatgtgtgagttcacccaga ggcctgctct 1800 cctcacacat tgtgtggttt ggggttaatg atggagggagacacctcttc atagacggca 1860 ggtgcccacc tttcagggag tctcccagca tgggcggatgccgggcatga gctgctgtaa 1920 actatttgtg gctgtgctgc ttgagtgacg tctctgtcgtgtgggtgcca agtgcttgtg 1980 tagaaactgt gttctgagcc cccttttctg gacaccaactgtgtcctgtg aatgtatcgc 2040 tactgtgagc tgttcccgcc tagccagggc catgtcttaggtgcagctgt gccacgggtc 2100 agctgagcca cagtcccaga accaagctct cggtgtctcgggccaccatc cgcccacctc 2160 gggctgaccc cacctcctcc atggacagtg tgagccccgggccgtgcatc ctgctcagtg 2220 tggcgtcagt gtcggggctg agccccttga gctgcttcagtgaatgtaca gtgcccggca 2280 cgagctgaac ctcatgtgtt ccactcccaa taaaaggttgacaggggctt ctccttcaaa 2340 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2371 471900 DNA Homo sapiens 47 gggggaatgt agggcagatg gtcccaacag gccagaggccactggtgtca ggcatcagca 60 gcttcctcct cagagtccag gtctgccgct gccttcgtgcccacggtctt cactccctgc 120 gccgggacca acaatgccct ggctcctgag ggctgcttttgacctgtgcg gggagcagcc 180 ccaacttggg gtggggtctt acaggcttct ggacctgccttttcctgaca attcaggtca 240 gagtctcaag tcccctggga ggggccccaa gcagctgtgaggcctgggtg ctcggagccc 300 actgaccgct agccttgtgc ttgtccccag cggcaagcctgcacgagctg tgtgctgcca 360 gagtgtcaga ggtccttcaa aacagagtgc acaggacggaggaagtgaag catgtggact 420 tctatgcttt ctcctactat tacgaccttg cagctggtgtgggcctcata gatgcggaga 480 agggaggcag cctggtggtg ggggacttcg agatcgcagccaagtacgtg tgtcggaccc 540 tggagacaca gccgcagagc agccccttct catgcatggacctcacctac gtcagcctgc 600 tactccagga gttcggcttt cccaggagca aagtgctgaagctcactcgg aaaattgaca 660 atgttgagac cagctgggct ctgggggcca tttttcattacatcgactcc ctgaacagac 720 agaagagtcc agcctcatag tggccgagcc atccctgtccccgtcagcag tgtctgtgtg 780 tctgcataaa ccctcctgtc ctggacgtga cttcatcctgaggagccaca gcacaggccg 840 tgctggcact ttctgcacac tggctctggg acttgcagaaggcctggtgc tgccctggca 900 tcagcctctt ccagtcacat ctggccagag ggctgtctggacctgggccc tgctcaatgc 960 cacctgtctg cctgggctcc aagtgggcag gaccaggacagaaccacagg cacacactga 1020 gggggcagtg tggctccctg cctgtcccat ccccatgccccgtccgcggg gctgtggctg 1080 ctgctgtgca tgtccctgcg atgggagtct tgtctcccagcctgtcagtt tcctccccag 1140 ggcagagctc cccttcctgc aagagtctgg gaggcggtgcaggctgtcct ggctgctctg 1200 gggaagccga gggacagcca taacaccccc gggacagtaggtctgggcgg caccactggg 1260 aactctggac ttgagtgtgt ttgctcttcc ttgggtatgaatgtgtgagt tcacccagag 1320 gcctgctctc ctcacacatt gtgtggtttg gggttaatgatggagggaga cacctcttca 1380 tagacggcag gtgcccacct ttcagggagt ctcccagcatgggcggatgc cgggcatgag 1440 ctgctgtaaa ctatttgtgg ctgtgctgct tgagtgacgtctctgtcgtg tgggtgccaa 1500 gtgcttgtgt agaaactgtg ttctgagccc ccttttctggacaccaactg tgtcctgtga 1560 atgtatcgct actgtgagct gttcccgcct agccagggccatgtcttagg tgcagctgtg 1620 ccacgggtca gctgagccac agtcccagaa ccaagctctcggtgtctcgg gccaccatcc 1680 gcccacctcg ggctgacccc acctcctcca tggacagtgtgagccccggg ccgtgcatcc 1740 tgctcagtgt ggcgtcagtg tcggggctga gccccttgagctgcttcagt gaatgtacag 1800 tgcccggcac gagctgaacc tcatgtgttc cactcccaataaaaggttga caggggcttc 1860 tccttcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa1900 48 2693 DNA Homo sapiens 48 gtggggtcgt atcccgcggg tggaggccggggtggcgccg gccggggcgg gggagcccaa 60 aagaccggct gccgcctgct ccccggaaaagggcactcgt ctccgtgggt gtggcggagc 120 gcgcggtgca tgcagccgca gcacggtccttggcaaacaa ggatgagaaa aatatccaac 180 cacgggagcc tgcgggtggc gaaggtggcataccccctgg ggctgtgtgt gggcgtgttc 240 atctatgttg cctacatcaa gtggcaccgggccaccgcca cccaggcctt cttcagcatc 300 accagggcag ccccgggggc ccggtggggtcagcaggccc acagccccct ggggacagct 360 gcagacgggc acgaggtctt ctacgggatcatgtttgatg caggaagcac tggcacccga 420 gtacacgtct tccagttcac ccggccccccagagaaactc ccacgttaac ccacgaaacc 480 ttcaaagcag tgaagccagg tctttctgcctatgctgatg atgttgaaaa gagcgctcag 540 ggaatccggg aactactgga tgttgctaaacaggacattc cgttcgactt ctggaaggcc 600 acccctctgg tcctcaaggc cacagctggcttacgcctgt tacctggaga aaaggcccag 660 aagttactgc agaaggtgaa agaagtatttaaagcatcgc ctttccttgt aggggatgac 720 tgtgtttcca tcatgaacgg aacagatgaaggcgtttcgg cgtggatcac catcaacttc 780 ctgacaggca gcttgaaaac tccaggagggagcagcgtgg gcatgctgga cttgggcgga 840 ggatccactc agatcgcctt cctgccacgcgtggagggca ccctgcaggc ctccccaccc 900 ggctacctga cggcactgcg gatgtttaacaggacctaca agctctattc ctacagctac 960 ctcgggctcg ggctgatgtc ggcacgcctggcgatcctgg gcggcgtgga ggggcagcct 1020 gctaaggatg gaaaggagtt ggtcagcccttgcttgtctc ccagtttcaa aggagagtgg 1080 gaacacgcag aagtcacgta cagggtttcagggcagaaag cagcggcaag cctgcacgag 1140 ctgtgtgctg ccagagtgtc agaggtccttcaaaacagag tgcacaggac ggaggaagtg 1200 aagcatgtgg acttctatgc tttctcctactattacgacc ttgcagctgg tgtgggcctc 1260 atagatgcgg agaagggagg cagcctggtggtgggggact tcgagatcgc agccaagtac 1320 gtgtgtcgga ccctggagac acagccgcagagcagcccct tctcatgcat ggacctcacc 1380 tacgtcagcc tgctactcca ggagttcggctttcccagga gcaaagtgct gaagctcact 1440 cggaaaattg acaatgttga gaccagctgggctctggggg ccatttttca ttacatcgac 1500 tccctgaaca gacagaagag tccagcctcatagtggccga gccatccctg tccccgtcag 1560 cagtgtctgt gtgtctgcat aaaccctcctgtcctggacg tgacttcatc ctgaggagcc 1620 acagcacagg ccgtgctggc actttctgcacactggctct gggacttgca gaaggcctgg 1680 tgctgccctg gcatcagcct cttccagtcacatctggcca gagggctgtc tggacctggg 1740 ccctgctcaa tgccacctgt ctgcctgggctccaagtggg caggaccagg acagaaccac 1800 aggcacacac tgagggggca gtgtggctccctgcctgtcc catccccatg ccccgtccgc 1860 ggggctgtgg ctgctgctgt gcatgtccctgcgatgggag tcttgtctcc cagcctgtca 1920 gtttcctccc cagggcagag ctccccttcctgcaagagtc tgggaggcgg tgcaggctgt 1980 cctggctgct ctggggaagc cgagggacagccataacacc cccgggacag taggtctggg 2040 cggcaccact gggaactctg gacttgagtgtgtttgctct tccttgggta tgaatgtgtg 2100 agttcaccca gaggcctgct ctcctcacacattgtgtggt ttggggttaa tgatggaggg 2160 agacacctct tcatagacgg caggtgcccacctttcaggg agtctcccag catgggcgga 2220 tgccgggcat gagctgctgt aaactatttgtggctgtgct gcttgagtga cgtctctgtc 2280 gtgtgggtgc caagtgcttg tgtagaaactgtgttctgag cccccttttc tggacaccaa 2340 ctgtgtcctg tgaatgtatc gctactgtgagctgttcccg cctagccagg gccatgtctt 2400 aggtgcagct gtgccacggg tcagctgagccacagtccca gaaccaagct ctcggtgtct 2460 cgggccacca tccgcccacc tcgggctgaccccacctcct ccatggacag tgtgagcccc 2520 gggccgtgca tcctgctcag tgtggcgtcagtgtcggggc tgagcccctt gagctgcttc 2580 agtgaatgta cagtgcccgg cacgagctgaacctcatgtg ttccactccc aataaaaggt 2640 tgacaggggc ttctccttca aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaa 2693 49 2294 DNA Homo sapiens 49 gtggggtcgtatcccgcggg tggaggccgg ggtggcgccg gccggggcgg gggagcccaa 60 aagaccggctgccgcctgct ccccggaaaa gggcactcgt ctccgtgggt gtggcggagc 120 gcgcggtgcatgagcgctca gggaatccgg gaactactgg atgttgctaa acaggacatt 180 ccgttcgacttctggaaggc cacccctctg gtcctcaagg ccacagctgg cttacgcctg 240 ttacctggagaaaaggccca gaagttactg cagaaggtga aagaagtatt taaagcatcg 300 cctttccttgtaggggatga ctgtgtttcc atcatgaacg gaacagatga aggcgtttcg 360 gcgtggatcaccatcaactt cctgacaggc agcttgaaaa ctccaggagg gagcagcgtg 420 ggcatgctggacttgggcgg aggatccact cagatcgcct tcctgccacg cgtggagggc 480 accctgcaggcctccccacc cggctacctg acggcactgc ggatgtttaa caggacctac 540 aagctctattcctacagcta cctcgggctc gggctgatgt cggcacgcct ggcgatcctg 600 ggcggcgtggaggggcagcc tgctaaggat ggaaaggagt tggtcagccc ttgcttgtct 660 cccagtttcaaaggagagtg ggaacacgca gaagtcacgt acagggtttc agggcagaaa 720 gcagcggcaagcctgcacga gctgtgtgct gccagagtgt cagaggtcct tcaaaacaga 780 gtgcacaggacggaggaagt gaagcatgtg gacttctatg ctttctccta ctattacgac 840 cttgcagctggtgtgggcct catagatgcg gagaagggag gcagcctggt ggtgggggac 900 ttcgagatcgcagccaagta cgtgtgtcgg accctggaga cacagccgca gagcagcccc 960 ttctcatgcatggacctcac ctacgtcagc ctgctactcc aggagttcgg ctttcccagg 1020 agcaaagtgctgaagctcac tcggaaaatt gacaatgttg agaccagctg ggctctgggg 1080 gccatttttcattacatcga ctccctgaac agacagaaga gtccagcctc atagtggccg 1140 agccatccctgtccccgtca gcagtgtctg tgtgtctgca taaaccctcc tgtcctggac 1200 gtgacttcatcctgaggagc cacagcacag gccgtgctgg cactttctgc acactggctc 1260 tgggacttgcagaaggcctg gtgctgccct ggcatcagcc tcttccagtc acatctggcc 1320 agagggctgtctggacctgg gccctgctca atgccacctg tctgcctggg ctccaagtgg 1380 gcaggaccaggacagaacca caggcacaca ctgagggggc agtgtggctc cctgcctgtc 1440 ccatccccatgccccgtccg cggggctgtg gctgctgctg tgcatgtccc tgcgatggga 1500 gtcttgtctcccagcctgtc agtttcctcc ccagggcaga gctccccttc ctgcaagagt 1560 ctgggaggcggtgcaggctg tcctggctgc tctggggaag ccgagggaca gccataacac 1620 ccccgggacagtaggtctgg gcggcaccac tgggaactct ggacttgagt gtgtttgctc 1680 ttccttgggtatgaatgtgt gagttcaccc agaggcctgc tctcctcaca cattgtgtgg 1740 tttggggttaatgatggagg gagacacctc ttcatagacg gcaggtgccc acctttcagg 1800 gagtctcccagcatgggcgg atgccgggca tgagctgctg taaactattt gtggctgtgc 1860 tgcttgagtgacgtctctgt cgtgtgggtg ccaagtgctt gtgtagaaac tgtgttctga 1920 gcccccttttctggacacca actgtgtcct gtgaatgtat cgctactgtg agctgttccc 1980 gcctagccagggccatgtct taggtgcagc tgtgccacgg gtcagctgag ccacagtccc 2040 agaaccaagctctcggtgtc tcgggccacc atccgcccac ctcgggctga ccccacctcc 2100 tccatggacagtgtgagccc cgggccgtgc atcctgctca gtgtggcgtc agtgtcgggg 2160 ctgagccccttgagctgctt cagtgaatgt acagtgcccg gcacgagctg aacctcatgt 2220 gttccactcccaataaaagg ttgacagggg cttctccttc aaaaaaaaaa aaaaaaaaaa 2280 aaaaaaaaaaaaaa 2294 50 2805 DNA Homo sapiens 50 gtggggtcgt atcccgcggg tggaggccggggtggcgccg gccggggcgg gggagcccaa 60 aagaccggct gccgcctgct ccccggaaaagggcactcgt ctccgtgggt gtggcggagc 120 gcgcggtgca tggaatgggc tatgtgaatgaaaaaaggta tccgttatga aacttccaga 180 aaaacgagct acatttttca gcagccgcagcacggtcctt ggcaaacaag gatgagaaaa 240 atatccaacc acgggagcct gcgggtggcgaaggtggcat accccctggg gctgtgtgtg 300 ggcgtgttca tctatgttgc ctacatcaagtggcaccggg ccaccgccac ccaggccttc 360 ttcagcatca ccagggcagc cccgggggcccggtggggtc agcaggccca cagccccctg 420 gggacagctg cagacgggca cgaggtcttctacgggatca tgtttgatgc aggaagcact 480 ggcacccgag tacacgtctt ccagttcacccggcccccca gagaaactcc cacgttaacc 540 cacgaaacct tcaaagcagt gaagccaggtctttctgcct atgctgatga tgttgaaaag 600 agcgctcagg gaatccggga actactggatgttgctaaac aggacattcc gttcgacttc 660 tggaaggcca cccctctggt cctcaaggccacagctggct tacgcctgtt acctggagaa 720 aaggcccaga agttactgca gaaggtgaaagaagtattta aagcatcgcc tttccttgta 780 ggggatgact gtgtttccat catgaacggaacagatgaag gcgtttcggc gtggatcacc 840 atcaacttcc tgacaggcag cttgaaaactccaggaggga gcagcgtggg catgctggac 900 ttgggcggag gatccactca gatcgccttcctgccacgcg tggagggcac cctgcaggcc 960 tccccacccg gctacctgac ggcactgcggatgtttaaca ggacctacaa gctctattcc 1020 tacagctacc tcgggctcgg gctgatgtcggcacgcctgg cgatcctggg cggcgtggag 1080 gggcagcctg ctaaggatgg aaaggagttggtcagccctt gcttgtctcc cagtttcaaa 1140 ggagagtggg aacacgcaga agtcacgtacagggtttcag ggcagaaagc agcggcaagc 1200 ctgcacgagc tgtgtgctgc cagagtgtcagaggtccttc aaaacagagt gcacaggacg 1260 gaggaagtga agcatgtgga cttctatgctttctcctact attacgacct tgcagctggt 1320 gtgggcctca tagatgcgga gaagggaggcagcctggtgg tgggggactt cgagatcgca 1380 gccaagtacg gtgggagtca cctggagcgtgaagggacgt gtctcatccc cagtgtgtcg 1440 gaccctggag acacagccgc agagcagccccttctcatgc atggacctca cctacgtcag 1500 cctgctactc caggagttcg gctttcccaggagcaaagtg ctgaagctca ctcggaaaat 1560 tgacaatgtt gagaccagct gggctctgggggccattttt cattacatcg actccctgaa 1620 cagacagaag agtccagcct catagtggccgagccatccc tgtccccgtc agcagtgtct 1680 gtgtgtctgc ataaaccctc ctgtcctggacgtgacttca tcctgaggag ccacagcaca 1740 ggccgtgctg gcactttctg cacactggctctgggacttg cagaaggcct ggtgctgccc 1800 tggcatcagc ctcttccagt cacatctggccagagggctg tctggacctg ggccctgctc 1860 aatgccacct gtctgcctgg gctccaagtgggcaggacca ggacagaacc acaggcacac 1920 actgaggggg cagtgtggct ccctgcctgtcccatcccca tgccccgtcc gcggggctgt 1980 ggctgctgct gtgcatgtcc ctgcgatgggagtcttgtct cccagcctgt cagtttcctc 2040 cccagggcag agctcccctt cctgcaagagtctgggaggc ggtgcaggct gtcctggctg 2100 ctctggggaa gccgagggac agccataacacccccgggac agtaggtctg ggcggcacca 2160 ctgggaactc tggacttgag tgtgtttgctcttccttggg tatgaatgtg tgagttcacc 2220 cagaggcctg ctctcctcac acattgtgtggtttggggtt aatgatggag ggagacacct 2280 cttcatagac ggcaggtgcc cacctttcagggagtctccc agcatgggcg gatgccgggc 2340 atgagctgct gtaaactatt tgtggctgtgctgcttgagt gacgtctctg tcgtgtgggt 2400 gccaagtgct tgtgtagaaa ctgtgttctgagcccccttt tctggacacc aactgtgtcc 2460 tgtgaatgta tcgctactgt gagctgttcccgcctagcca gggccatgtc ttaggtgcag 2520 ctgtgccacg ggtcagctga gccacagtcccagaaccaag ctctcggtgt ctcgggccac 2580 catccgccca cctcgggctg accccacctcctccatggac agtgtgagcc ccgggccgtg 2640 catcctgctc agtgtggcgt cagtgtcggggctgagcccc ttgagctgct tcagtgaatg 2700 tacagtgccc ggcacgagct gaacctcatgtgttccactc ccaataaaag gttgacaggg 2760 gcttctcctt caaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaa 2805 51 2497 DNA Homo sapiens 51 tgctgccttc ctgtccaggctccattaggg ctccctggat gtctgggctc cgctgaccgg 60 tggggggcaa gtccagtgttttgggccaca gggcgggcca aatagcaggg gcctgtagcc 120 ccagggggcc ccagccctgctgcgttcatt cattcagccc gcttttgcgg acccgccccc 180 agtctgcgtc agggacagacccctccgccc tggatgagcc cggcggggaa gcttcgcgta 240 gcagtttctg ggaaacagaaactcccacgt taacccacga aaccttcaaa gcagtgaagc 300 caggtctttc tgcctatgctgatgatgttg aaaagagcgc tcagggaatc cgggaactac 360 tggatgttgc taaacaggacattccgttcg acttctggaa ggccacccct ctggtcctca 420 aggccacagc tggcttacgcctgttacctg gagaaaaggc ccagaagtta ctgcagaagg 480 tgaaagaagt atttaaagcatcgcctttcc ttgtagggga tgactgtgtt tccatcatga 540 acggaacaga tgaaggcgtttcggcgtgga tcaccatcaa cttcctgaca ggcagcttga 600 aaactccagg agggagcagcgtgggcatgc tggacttggg cggaggatcc actcagatcg 660 ccttcctgcc acgcgtggagggcaccctgc aggcctcccc acccggctac ctgacggcac 720 tgcggatgtt taacaggacctacaagctct attcctacag ctacctcggg ctcgggctga 780 tgtcggcacg cctggcgatcctgggcggcg tggaggggca gcctgctaag gatggaaagg 840 agttggtcag cccttgcttgtctcccagtt tcaaaggaga gtgggaacac gcagaagtca 900 cgtacagggt ttcagggcagaaagcagcgg caagcctgca cgagctgtgt gctgccagag 960 tgtcagaggt ccttcaaaacagagtgcaca ggacggagga agtgaagcat gtggacttct 1020 atgctttctc ctactattacgaccttgcag ctggtgtggg cctcatagat gcggagaagg 1080 gaggcagcct ggtggtgggggacttcgaga tcgcagccaa gtacgtgtgt cggaccctgg 1140 agacacagcc gcagagcagccccttctcat gcatggacct cacctacgtc agcctgctac 1200 tccaggagtt cggctttcccaggagcaaag tgctgaagct cactcggaaa attgacaatg 1260 ttgagaccag ctgggctctgggggccattt ttcattacat cgactccctg aacagacaga 1320 agagtccagc ctcatagtggccgagccatc cctgtccccg tcagcagtgt ctgtgtgtct 1380 gcataaaccc tcctgtcctggacgtgactt catcctgagg agccacagca caggccgtgc 1440 tggcactttc tgcacactggctctgggact tgcagaaggc ctggtgctgc cctggcatca 1500 gcctcttcca gtcacatctggccagagggc tgtctggacc tgggccctgc tcaatgccac 1560 ctgtctgcct gggctccaagtgggcaggac caggacagaa ccacaggcac acactgaggg 1620 ggcagtgtgg ctccctgcctgtcccatccc catgccccgt ccgcggggct gtggctgctg 1680 ctgtgcatgt ccctgcgatgggagtcttgt ctcccagcct gtcagtttcc tccccagggc 1740 agagctcccc ttcctgcaagagtctgggag gcggtgcagg ctgtcctggc tgctctgggg 1800 aagccgaggg acagccataacacccccggg acagtaggtc tgggcggcac cactgggaac 1860 tctggacttg agtgtgtttgctcttccttg ggtatgaatg tgtgagttca cccagaggcc 1920 tgctctcctc acacattgtgtggtttgggg ttaatgatgg agggagacac ctcttcatag 1980 acggcaggtg cccacctttcagggagtctc ccagcatggg cggatgccgg gcatgagctg 2040 ctgtaaacta tttgtggctgtgctgcttga gtgacgtctc tgtcgtgtgg gtgccaagtg 2100 cttgtgtaga aactgtgttctgagccccct tttctggaca ccaactgtgt cctgtgaatg 2160 tatcgctact gtgagctgttcccgcctagc cagggccatg tcttaggtgc agctgtgcca 2220 cgggtcagct gagccacagtcccagaacca agctctcggt gtctcgggcc accatccgcc 2280 cacctcgggc tgaccccacctcctccatgg acagtgtgag ccccgggccg tgcatcctgc 2340 tcagtgtggc gtcagtgtcggggctgagcc ccttgagctg cttcagtgaa tgtacagtgc 2400 ccggcacgag ctgaacctcatgtgttccac tcccaataaa aggttgacag gggcttctcc 2460 ttcaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaa 2497 52 2762 DNA Homo sapiens 52 gtggggtcgtatcccgcggg tggaggccgg ggtggcgccg gccggggcgg gggagcccaa 60 aagaccggctgccgcctgct ccccggaaaa gggcactcgt ctccgtgggt gtggcggagc 120 gcgcggtgcatggaatgggc tatgtgaatg aaaaaaggta tccgttatga aacttccaga 180 aaaacgagctacatttttca gcagccgcag cacggtcctt ggcaaacaag gatgagaaaa 240 atatccaaccacgggagcct gcgggtggcg aaggtggcat accccctggg gctgtgtgtg 300 ggcgtgttcatctatgttgc ctacatcaag tggcaccggg ccaccgccac ccaggccttc 360 ttcagcatcaccagggcagc cccgggggcc cggtggggtc agcaggccca cagccccctg 420 gggacagctgcagacgggca cgaggtcttc tacgggatca tgtttgatgc aggaagcact 480 ggcacccgagtacacgtctt ccagttcacc cggcccccca gagaaactcc cacgttaacc 540 cacgaaaccttcaaagcagt gaagccaggt ctttctgcct atgctgatga tgttgaaaag 600 agcgctcagggaatccggga actactggat gttgctaaac aggacattcc gttcgacttc 660 tggaaggccacccctctggt cctcaaggcc acagctggct tacgcctgtt acctggagaa 720 aaggcccagaagttactgca gaaggtgaaa gaagtattta aagcatcgcc tttccttgta 780 ggggatgactgtgtttccat catgaacgga acagatgaag gcgtttcggc gtggatcacc 840 atcaacttcctgacaggcag cttgaaaact ccaggaggga gcagcgtggg catgctggac 900 ttgggcggaggatccactca gatcgccttc ctgccacgcg tggagggcac cctgcaggcc 960 tccccacccggctacctgac ggcactgcgg atgtttaaca ggacctacaa gctctattcc 1020 tacagctacctcgggctcgg gctgatgtcg gcacgcctgg cgatcctggg cggcgtggag 1080 gggcagcctgctaaggatgg aaaggagttg gtcagccctt gcttgtctcc cagtttcaaa 1140 ggagagtgggaacacgcaga agtcacgtac agggtttcag ggcagaaagc agcggcaagc 1200 ctgcacgagctgtgtgctgc cagagtgtca gaggtccttc aaaacagagt gcacaggacg 1260 gaggaagtgaagcatgtgga cttctatgct ttctcctact attacgacct tgcagctggt 1320 gtgggcctcatagatgcgga gaagggaggc agcctggtgg tgggggactt cgagatcgca 1380 gccaagtacgtgtgtcggac cctggagaca cagccgcaga gcagcccctt ctcatgcatg 1440 gacctcacctacgtcagcct gctactccag gagttcggct ttcccaggag caaagtgctg 1500 aagctcactcggaaaattga caatgttgag accagctggg ctctgggggc catttttcat 1560 tacatcgactccctgaacag acagaagagt ccagcctcat agtggccgag ccatccctgt 1620 ccccgtcagcagtgtctgtg tgtctgcata aaccctcctg tcctggacgt gacttcatcc 1680 tgaggagccacagcacaggc cgtgctggca ctttctgcac actggctctg ggacttgcag 1740 aaggcctggtgctgccctgg catcagcctc ttccagtcac atctggccag agggctgtct 1800 ggacctgggccctgctcaat gccacctgtc tgcctgggct ccaagtgggc aggaccagga 1860 cagaaccacaggcacacact gagggggcag tgtggctccc tgcctgtccc atccccatgc 1920 cccgtccgcggggctgtggc tgctgctgtg catgtccctg cgatgggagt cttgtctccc 1980 agcctgtcagtttcctcccc agggcagagc tccccttcct gcaagagtct gggaggcggt 2040 gcaggctgtcctggctgctc tggggaagcc gagggacagc cataacaccc ccgggacagt 2100 aggtctgggcggcaccactg ggaactctgg acttgagtgt gtttgctctt ccttgggtat 2160 gaatgtgtgagttcacccag aggcctgctc tcctcacaca ttgtgtggtt tggggttaat 2220 gatggagggagacacctctt catagacggc aggtgcccac ctttcaggga gtctcccagc 2280 atgggcggatgccgggcatg agctgctgta aactatttgt ggctgtgctg cttgagtgac 2340 gtctctgtcgtgtgggtgcc aagtgcttgt gtagaaactg tgttctgagc ccccttttct 2400 ggacaccaactgtgtcctgt gaatgtatcg ctactgtgag ctgttcccgc ctagccaggg 2460 ccatgtcttaggtgcagctg tgccacgggt cagctgagcc acagtcccag aaccaagctc 2520 tcggtgtctcgggccaccat ccgcccacct cgggctgacc ccacctcctc catggacagt 2580 gtgagccccgggccgtgcat cctgctcagt gtggcgtcag tgtcggggct gagccccttg 2640 agctgcttcagtgaatgtac agtgcccggc acgagctgaa cctcatgtgt tccactccca 2700 ataaaaggttgacaggggct tctccttcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760 aa 2762 531588 DNA Homo sapiens 53 gtggggtcgt atcccgcggg tggaggccgg ggtggcgccggccggggcgg gggagcccaa 60 aagaccggct gccgcctgct ccccggaaaa gggcactcgtctccgtgggt gtggcggagc 120 gcgcggtgca tggaatgggc tatgtgaatg aaaaaaggtatccgttatga aacttccaga 180 aaaacgagct acatttttca gcagccgcag cacggtccttggcaaacaag gatgagaaaa 240 atatccaacc acgggagcct gcgggtggcg aaggtggcataccccctggg gctgtgtgtg 300 ggcgtgttca tctatgttgc ctacatcaag tggcaccgggccaccgccac ccaggccttc 360 ttcagcatca ccagggcagc cccgggggcc cggtggggtcagcaggccca cagccccctg 420 gggacagctg cagacgggca cgaggtcttc tacgggatcatgtttgatgc aggaagcact 480 ggcacccgag tacacgtctt ccagttcacc cggccccccagagaaactcc cacgttaacc 540 cacgaaacct tcaaagcagt gaagccaggt ctttctgcctatgctgatga tgttgaaaag 600 agcgctcagg gaatccggga actactggat gttgctaaacaggacattcc gttcgacttc 660 tggaaggcca cccctctggt cctcaaggcc acagctggcttacgcctgtt acctggagaa 720 aaggcccaga agttactgca gaaggtgaaa gaagtatttaaagcatcgcc tttccttgta 780 ggggatgact gtgtttccat catgaacgga acagatgaaggcgtttcggc gtggatcacc 840 atcaacttcc tgacaggcag cttgaaaact ccaggagggagcagcgtggg catgctggac 900 ttgggcggag gatccactca gatcgccttc ctgccacgcgtggagggcac cctgcaggcc 960 tccccacccg gctacctgac ggcactgcgg atgtttaacaggacctacaa gctctattcc 1020 tacagctacc tcgggctcgg gctgatgtcg gcacgcctggcgatcctggg cggcgtggag 1080 gggcagcctg ctaaggatgg aaaggagttg gtcagcccttgcttgtctcc cagtttcaaa 1140 ggagagtggg aacacgcaga agtcacgtac agggtttcagggcagaaagc agcggcaagc 1200 ctgcacgagc tgtgtgctgc cagagtgtca gaggtccttcaaaacagagt gcacaggacg 1260 gaggaagtga agcatgtgga cttctatgct ttctcctactattacgacct tgcagctggt 1320 gtgggcctca tagatgcgga gaagggaggc agcctggtggtgggggactt cgagatcgca 1380 gccaagtacg aaagcttagg ttttatgcag cttgacacctgggagaggtg tccagggtct 1440 tagatgtaga aatgtggctt gggagacatt ggcccatgaccctaacacca ggcgtgcaaa 1500 tccccttgga gcccagaccc agtggcagcc ggggttgccgcccacctgcc tgtgctgtgc 1560 actgggtgcc ttctgcgctc agctgcgg 1588 54 2882DNA Homo sapiens 54 gtggggtcgt atcccgcggg tggaggccgg ggtggcgccggccggggcgg gggagcccaa 60 aagaccggct gccgcctgct ccccggaaaa gggcactcgtctccgtgggt gtggcggagc 120 gcgcggtgca tggaatgggc tatgtgaatg aaaaaaggtatccgttatga aacttccaga 180 aaaacgagct acatttttca gcagccgcag cacggtccttggcaaacaag gatgagaaaa 240 atatccaacc acgggagcct gcgggtggcg aaggtggcataccccctggg gctgtgtgtg 300 ggcgtgttca tctatgttgc ctacatcaag tggcaccgggccaccgccac ccaggccttc 360 ttcagcatca ccagggcagc cccgggggcc cggtggggtcagcaggccca cagccccctg 420 gggacagctg cagacgggca cgaggtcttc tacgggatcatgtttgatgc aggaagcact 480 ggcacccgag tacacgtctt ccagttcacc cggccccccagagaaactcc cacgttaacc 540 cacgaaacct tcaaagcagt gaagccaggt ctttctgcctatgctgatga tgttgaaaag 600 agcgctcagg gaatccggga actactggat gttgctaaacaggacattcc gttcgacttc 660 tggaaggcca cccctctggt cctcaaggcc acagctggcttacgcctgtt acctggagaa 720 aaggcccaga agttactgca gaaggtgaaa gaagtatttaaagcatcgcc tttccttgta 780 ggggatgact gtgtttccat catgaacgga acagatgaaggcgtttcggc gtggatcacc 840 atcaacttcc tgacaggcag cttgaaaact ccaggagggagcagcgtggg catgctggac 900 ttgggcggag gatccactca gatcgccttc ctgccacgcgtggagggcac cctgcaggcc 960 tccccacccg gctacctgac ggcactgcgg atgtttaacaggacctacaa gctctattcc 1020 tacagctacc tcgggctcgg gctgatgtcg gcacgcctggcgatcctggg cggcgtggag 1080 gggcagcctg ctaaggatgg aaaggagttg gtcagcccttgcttgtctcc cagtttcaaa 1140 ggagagtggg aacacgcaga agtcacgtac agggtttcagggcagaaagc agcggcaagc 1200 ctgcacgagc tgtgtgctgc cagagtgtca gaggtccttcaaaacagagt gcacaggacg 1260 gaggaagtga agcatgtgga cttctatgct ttctcctactattacgacct tgcagctggt 1320 gtgggcctca taggaggcca gaagcagatt tgagaaggcgtctcatcctg agtaggaaaa 1380 gatcagttct ttgagccctt cagtaaaacc tcgtggctggtgacttgctg ttgattccag 1440 ttcctgctgg aagatgcgga gaagggaggc agcctggtggtgggggactt cgagatcgca 1500 gccaagtacg tgtgtcggac cctggagaca cagccgcagagcagcccctt ctcatgcatg 1560 gacctcacct acgtcagcct gctactccag gagttcggctttcccaggag caaagtgctg 1620 aagctcactc ggaaaattga caatgttgag accagctgggctctgggggc catttttcat 1680 tacatcgact ccctgaacag acagaagagt ccagcctcatagtggccgag ccatccctgt 1740 ccccgtcagc agtgtctgtg tgtctgcata aaccctcctgtcctggacgt gacttcatcc 1800 tgaggagcca cagcacaggc cgtgctggca ctttctgcacactggctctg ggacttgcag 1860 aaggcctggt gctgccctgg catcagcctc ttccagtcacatctggccag agggctgtct 1920 ggacctgggc cctgctcaat gccacctgtc tgcctgggctccaagtgggc aggaccagga 1980 cagaaccaca ggcacacact gagggggcag tgtggctccctgcctgtccc atccccatgc 2040 cccgtccgcg gggctgtggc tgctgctgtg catgtccctgcgatgggagt cttgtctccc 2100 agcctgtcag tttcctcccc agggcagagc tccccttcctgcaagagtct gggaggcggt 2160 gcaggctgtc ctggctgctc tggggaagcc gagggacagccataacaccc ccgggacagt 2220 aggtctgggc ggcaccactg ggaactctgg acttgagtgtgtttgctctt ccttgggtat 2280 gaatgtgtga gttcacccag aggcctgctc tcctcacacattgtgtggtt tggggttaat 2340 gatggaggga gacacctctt catagacggc aggtgcccacctttcaggga gtctcccagc 2400 atgggcggat gccgggcatg agctgctgta aactatttgtggctgtgctg cttgagtgac 2460 gtctctgtcg tgtgggtgcc aagtgcttgt gtagaaactgtgttctgagc ccccttttct 2520 ggacaccaac tgtgtcctgt gaatgtatcg ctactgtgagctgttcccgc ctagccaggg 2580 ccatgtctta ggtgcagctg tgccacgggt cagctgagccacagtcccag aaccaagctc 2640 tcggtgtctc gggccaccat ccgcccacct cgggctgaccccacctcctc catggacagt 2700 gtgagccccg ggccgtgcat cctgctcagt gtggcgtcagtgtcggggct gagccccttg 2760 agctgcttca gtgaatgtac agtgcccggc acgagctgaacctcatgtgt tccactccca 2820 ataaaaggtt gacaggggct tctccttcaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2880 aa 2882

What is claimed is:
 1. An isolated polynucleotide encoding an apyraseand/or NDPase and comprising a nucleotide sequence having at least about80% sequence identity to a human polynucleotide selected from the groupconsisting of: (a) a polynucleotide having the nucleotide sequence ofSEQ ID NO. 2; and (b) a polynucleotide having the protein codingnucleotide sequence of the polynucleotide sequence of (a).
 2. Anisolated polynucleotide encoding an apyrase and comprising a nucleotidesequence having at least about 90% sequence identity to a polynucleotideselected from the group consisting of: (a) a polynucleotide having thenucleotide sequence of SEQ ID NO. 2; and (b) a polynucleotide having theprotein coding nucleotide sequence of the polynucleotide sequence of(a).
 3. An isolated polynucleotide encoding a polypeptide with apyraseand/or NDPase activity, comprising a polynucleotide selected from thegroup consisting of: (a) polynucleotides that encode the mature proteincoding amino acid sequence of SEQ ID NO.
 3. 4. An isolatedpolynucleotide encoding a polypeptide with apyrase and/or NDPaseactivity that hybridizes under stringent conditions to the complement ofa polynucleotide of SEQ ID NO.
 2. 5. The polynucleotide of any one ofclaims 1 through 4 which is a DNA.
 6. The DNA of claim 5 which is awholly or partially chemically synthesized DNA molecule.
 7. Ananti-sense polynucleotide which specifically hybridizes with thecomplement of the polynucleotide of claim
 4. 8. The polynucleotide ofclaim 1 which comprises the nucleotide sequence of SEQ ID NO. 2 or 24 orthe mature protein coding portions thereof.
 9. An isolatedpolynucleotide which comprises a complement of the polynucleotide ofclaim
 1. 10. An expression vector comprising the DNA of claim
 5. 11. Ahost cell comprising the DNA of claim
 5. 12. A host cell geneticallyengineered to express the DNA of claim
 5. 13. An isolated humanpolypeptide with apyrase and/or NDPase activity comprising: (a) themature protein coding sequence of SEQ ID NO. 3; or (b) an amino acidsequence having at least about 80% sequence identity to SEQ ID NO. 3.14. An isolated polypeptide with apyrase and/or NDPase activitycomprising: (a) the CD39-like protein coding sequence of SEQ ID NO. 3;or (b) an amino acid sequence having at least about 90% sequenceidentity to SEQ ID NO.
 3. 15. The polypeptide of claim 3 or 14 whichcomprises the amino acid sequence of SEQ ID NO. 3 or 25 or the matureprotein portions thereof.
 16. The polypeptide of claim 13 or 14 whereinthe polypeptide comprises at least one amino acid substitution selectedfrom the group consisting of: D168→T, S170→Q and L175→F.
 17. Thepolypeptide of claim 16 comprising a polypeptide having the amino acidsequence set forth in SEQ ID NO.
 7. 18. A method for producing aCD39-like polypeptide comprising the steps of: (a) growing a culture ofhost cells according to claim 11 or 12 under conditions permittingexpression of a CD39-like polypeptide; and (b) isolating the CD39-likepolypeptide from the host cell or its growth medium.
 19. The method ofclaim 18 wherein the host cell is E. coli.
 20. A composition comprisingthe polypeptide of claim 13, 14 or 15 and a pharmaceutically acceptablecarrier.
 21. An antibody specifically immunoreactive with a polypeptideencoded by the polynucleotide according to claim
 1. 22. The antibodyaccording to claim 21 which is a monoclonal antibody.
 23. A hybridomawhich secretes the antibody according to claim
 22. 24. An anti-idiotypeantibody specifically immunoreactive with the antibody according toclaim
 22. 25. A method for detecting a polynucleotide of claim 1, 2 or 3in a sample comprising the steps of: (a) contacting the sample with acompound that binds to and forms a complex with the polynucleotide for aperiod sufficient to detect the complex; and (b) detecting the complexso that if a complex is detected, a polynucleotide of claim 1, 2 or 3 isdetected.
 26. A method for detecting a polynucleotide of claim 1, 2 or 3in a sample comprising the steps of: (a) contacting the sample understringent hybridization conditions with nucleic acid primers that annealto a polynucleotide of claim 1, 2 or 3 under such conditions; and (b)amplifying the polynucleotides of claim 1, 2 or 3 so that if apolynucleotide is amplified, a polynucleotide of claim 1, 2 or 3 isdetected.
 27. The method of claim 26 wherein the polynucleotide is anRNA molecule that encodes a polypeptide of claim 13 or 14, and themethod further comprises reverse transcribing an annealed RNA moleculeinto a cDNA polynucleotide.
 28. A method for detecting a polypeptide ofclaim 13 or 14 in a sample comprising: (a) contacting the sample with acompound that binds to and forms a complex with the polypeptide for aperiod sufficient to detect the complex; and (b) detecting the complexso that if a complex is detected, a polypeptide of claim 13 or 14 isdetected.
 29. A method for identifying a compound that binds to apolypeptide of claim 13 or 14 comprising: (a) contacting a compound witha polypeptide of claim 13 or 14 for a time sufficient to form apolypeptide/compound complex; and (b) detecting the complex so that if apolypeptide/compound complex is detected, a compound that binds to apolypeptide of claim 13 or 14 is detected.
 30. A method for identifyinga compound that binds to a polypeptide of claim 13 or 14 comprising: (a)contacting a compound with a polypeptide of claim 13 or 14, in a cell,for a time sufficient to form a polypeptide/compound complex, whereinthe complex drives expression of a reporter gene sequence in the cell,and (b) detecting the complex by detecting reporter gene sequenceexpression so that if a polypeptide/compound complex is detected, acompound that binds to a polypeptide of claim 13 or 14 is identified.31. A method of identifying a modulator compound of a CD39-likepolypeptide with apyrase activity comprising the steps of: (a)contacting the CD39-like polypeptide encoded by the polynucleotide ofclaim 1, 2 or 3 with a substrate in the presence and absence of a testcompound; (b) comparing apyrase activity of the CD39-like polypeptide inthe presence and absence of the test compound; and (c) identifying thetest compound as a modulator compound when biological activity of theCD39-like polypeptide is increased or decreased in the presence of thetest compound.
 32. A method of identifying a modulator compound of aCD39-like polypeptide with NDPase activity comprising the steps of: (a)contacting the CD39-like polypeptide encoded by the polynucleotide ofclaim 1, 2 or 3 with a substrate in the presence and absence of a testcompound; (b) comparing NDPase activity of the CD39-like polypeptide inthe presence and absence of the test compound; and (c) identifying thetest compound as a modulator compound when biological activity of theCD39-like polypeptide is increased or decreased in the presence of thetest compound.
 33. A chimeric polypeptide comprising one or more domainsof a CD39-like polypeptide fused to one or more domains of heterologouspeptide or polypeptide, e.g., an immunoglobulin constant region.
 34. Amethod of treatment comprising administering to a mammalian subject inneed thereof a therapeutic amount of a composition comprising apolypeptide of claim 13 or 14 and a pharmaceutically acceptable carrier.35. A method of treatment comprising administering to a mammaliansubject in need thereof a therapeutic amount of a composition comprisingan antibody that specifically binds to a polypeptide of claim 13 or 14and a pharmaceutically acceptable carrier.
 36. A method of inhibitingplatelet function comprising administering the polypeptide of claim 13or 14 to a medium comprising platelets.
 37. A method of treatingthrombotic diseases comprising administering a therapeutic amount of thepolypeptide of claim 13 or 14 to a mammalian subject in need thereof.38. A method of hydrolyzing nucleotidediphosphates comprisingadministering the polypeptide of claim 13 or 14 to a medium comprisingnucleotide diphosphates.
 39. A method of inhibiting platelet aggregationin a mammalian subject comprising the step of reducing the ratio ofADP:ATP in said mammalian subject to a less than normal ratio.
 40. Themethod of claim 39 wherein said ratio is reduced by administration ofCD39-L4 having the sequence set forth in SEQ ID NO: 3 or a polypeptidehaving NDPase activity and at least about 90% sequence identity to SEQID NO:
 3. 41. The method of claim 39 wherein said ratio is reduced byadministration of CD39-L66 having the sequence set forth in SEQ ID NO:25 or a polypeptide having NDPase activity and at least about 90%sequence identity to SEQ ID NO:
 25. 42. The method of claim 39 whereinsaid ratio is reduced by administration of CD39-L2 having the sequenceset forth in SEQ ID NO: 27 or a polypeptide having NDPase activity andat least about 90% sequence identity to SEQ ID NO:
 27. 43. The method ofany one of claims 39 through 42 wherein the ratio of ADP:ATP is reducedsystemically in circulation.
 44. The method of any one of claims 39through 42 wherein the ratio of ADP:ATP is reduced locally within anarea selected from the group consisting of heart, brain, kidney, lungand limbs.
 45. The method of any one of claims 39 through 42 wherein theratio of ADP:ATP is reduced without significantly affecting ATP levels.46. A method of identifying a compound capable of reducing the ratio ofADP:ATP to a less than normal ratio comprising the steps of: (a)determining apyrase activity of said compound on ATP; (b) determiningapyrase activity of said compounds on ADP; and (c) selecting a compoundthat has greater activity with respect to ADP compared to ATP.